THE 


MICROTOMIST'S  VADE-MECUM 


A  HANDBOOK  OP  THE  METHODS  OF 
MICROSCOPIC  ANATOMY 


BY 

ARTHUR    BOLLES    LEE 

\\ 

UNIVERSITY 


•*& 


FIFTH  EDITION 


PHILADELPHIA 
P.    BLAKISTON'S    SON    AND    CO. 

1012  WALNUT   STREET 

1900 


Printed  in  Great  Britain. 


to 


PREFACE. 


THE  present  edition  is  the  outcome  of  a  double  revision  of 
the  text  of  the  last  edition.  I  revised  that  text  very  thoroughly 
in  1897  in  conjunction  with  Prof.  Paul  Mayer,  in  view  of  the 
preparation  of  the  German  edition  that  has  since  appeared.* 
But  I  have  not  contented  myself  with  taking  the  text  thus 
settled  as  the  basis  of  the  present  edition  and  merely  adding 
such  newer  methods  as  have  appeared  up  to  date. 

For  on  the  one  hand  the  space  taken  up  by  description  of 
recent  processes  has  turned  out  to  be  so  considerable  as  to  call 
for  a  very  severe  condensation  of  the  text  throughout,  in  order 
to  keep  the  whole  within  the  old  limits  of  size — limits  which, 
it  seems  to  me,  cannot  be  much  increased  without  impairing 
the  handiness  of  the  volume  :  so  that  a  re-revision  seemed 
necessary  on  that  head.  And  on  the  other  hand  it  also 
seemed  to  me  desirable  to  amplify  even  more  fully  than  was 
done  in  the  last  edition  many  of  the  introductory  portions  of 
the  different  chapters,  and  to  discuss  still  more  fully  the 
principles  of  some  of  the  methods  described. 

Both  of  these  points  have  been  carried  out.  Phraseology 
has  been  curtailed  throughout  to  the  utmost  limits  consistent 
with  clearness.  Rejection  of  superfluous  methods  has  been 
carried  out  wholesale,  even  to  the  extent  of  suppression  of  a 
large  number  of  the  references  to  rejected  methods  that  were 
given  in  the  last  edition.  The  former  chapter  on  Hardening 
Agents  has  been  suppressed  and  its  contents  incorporated 

*  Grundzilge  der  mikroskopischen  Technikfilr  Zoologen  und  Anatomen, 
von  A.  B.  Lee  und  Paul  Mayer;  Berlin,  Friedlaender  und  Sohn,  1898.  I 
refer  to  this  edition  under  the  abbreviated  title  of  Grundzilge. 


vi  PREFACE. 

with  the  two  chapters  on  Fixing  and  Hardening  Agents,  thus 
saving  much  unnecessary  repetition.  And  on  the  other  hand 
there  has  been  added  a  great  deal  of  new  explanatory  matter 
concerning  such  subjects  as  the  theory  of  fixation,  microtome 
knives  and  knife-position,  serial  section  methods,  and  the  like  : 
the  chapter  on  Cytological  Methods  has  been  for  the  most 
part  rewritten :  and  throughout  Part  II  numerous  new 
examples  have  taken  the  place  of  former  ones,  or  have  been 
added  to  them. 

The  amount  of  new  matter  thus  brought  into  the  book  is 
very  considerable  ;  but  notwithstanding  this  the  size  of  the 
volume  has  not  been  increased,  and  I  venture  to  hope  that  the 
reader  will  find  that  the  labour  that  has  been  expended  on  it 
has  not  been  expended  in  vain. 


COLOGNY,  GENEVA,  SWITZERLAND; 
May,  1900. 


PREFACE 


TO   THE 


FOURTH    EDITION 


THK  short  period  of  three  years  that  has  elapsed  since  the 
publication  of  the  last  edition  of  this  work  has  not  brought 
with  it  any  radical  change  in  the  methods  of  histological 
research.  Such  progress  as  has  been  realised  has  consisted 
rather  in  improvements  in  the  detail  of  already-established 
methods  than  in  the  introduction  of  new  methods  or  new 
reagents.  Nevertheless,  the  present  edition  has  undergone 
a  most  thorough  revision — a  revision  indeed  so  thorough  as 
to  amount  to  extensive  re-writing  in  many  parts. 

It  has  seemed  to  me  advisable  in  the  interest  of  the  be- 
ginner, and  indeed  in  the  interest  of  readers  who  are  not 
beginners  at  all,  to  enter  more  fully  than  was  hitherto  done 
into  the  detail  of  the  more  important  processes,  to  explain 
more  fully  the  principles  on  which  they  are  founded,  and  to 
add  in  many  cases  a  critical  estimate  of  their  rationality  and 
practical  value.  In  consequence  of  this  re-writing,  and  in 
spite  of  strenuous  efforts  to  keep  down  the  bulk  of  the  work, 
it  has  turned  out  to  be  considerably  increased.  I  regret, 
however,  this  increase  the  less,  in  so  far  as  it  is  due  rather 
to  the  ampler  treatment  that  has  been  accorded  to  the  more 
valuable  methods  than  to  increase  in  the  number  of  processes 
described.  The  number  of  new  processes  described  is  in  fact 


vin  PEE  FACE. 

a  smaller  one  than  I  have  had  to  deal  with  in  the  preparation 
of  any  edition  since  the  first. 

The  classification  of  the  various  methods  has  received  most 
careful  attention,  and  has  been  in  many  cases  greatly  simpli- 
fied, whilst  at  the  same  time  a  large  number  of  .superfluous 
processes  have  been  rejected.  Advice  to  the  beginner  con- 
cerning the  choice  of  methods  has  been  given  wherever 
practicable,  and  I  think  that  notwithstanding  the  abundance 
and  complexity  of  the  matters  treated  of,  there  can  hardly 
be  any  risk  that  the  student  may  be  unable  to  see  the  wood 
for  the  trees. 

The  chapters  treating  of  Staining  and  of  the  Carmine  and 
Haematein  stains  have  had  the  great  advantage  of  revision 
by  Dr.  Paul  Mayer,  who,  it  is  superfluous  to  remind  the 
reader,  has  made  a  speciality  of  this  subject,  with  results 
brilliant  alike  in  theory  and  in  practice.  Not  indeed  that 
the  present  English  text  has  been  directly  revised  by  him, 
but  that  it  has  been  prepared  from  a  recent  text  so  revised. 
Dr.  Mayer  was  good  enough  to  revise  most  carefully  the 
three  corresponding  chapters  prepared  by  me  for  the  recent 
new  edition  of  the  Traite  des  Methodes  Techniques  de  I'Ana- 
tomie  Microscopique  (LEE  et  HENNEGUY),  and  in  the  prepara- 
tion of  the  present  English  text  I  have  closely  followed  the 
chapters  so  revised. 

No  less  obligation  have  I  to  express  to  Professor  van 
Grehuchten,  who  with  great  kindness  has  thoroughly  revised 
for  me  the  three  chapters  entitled  Neurological  Methods.  It 
occurred  to  me  that  my  treatment  of  this  complicated  subject 
could  not  but  gain  greatly  by  the  advice  of  an  observer  who 
is  not  only  one  of  the  foremost  of  the  new  school  of  neuro- 
logists but  at  the  same  time  an  instructed  and  capable 
cytologist,  and  therefore  likely  to  sympathise  with  my  feeling 
that  it  would  be  much  to  be  deplored  that  the  study  of 
nervous  anatomy  should  degenerate  into  a  mere  study  of 
topographical  relations,  to  the  neglect  of  the  inner  mechanism 
of  nervous  elements.  By  Professor  van  Grehuchten's  advice 


PREFACE.  ix 

I  have  entirely  re-arranged  the  contents  of  these  three 
chapters  according  to  a  scheme  worked  out  by  him,  thereby 
effecting  a  great  gain  in  clearness  of  exposition.  I  cannot 
Imt  acknowledge  that  the  arrangement  adopted  in  previous 
editions  resulted  in  something  like  a  chaos  ;  whilst  the  new 
arrangement  may,  I  think,  fairly  claim  to  be  natural,  logical, 
and  easily  comprehensible.  By  his  advice,  too,  I  have  entirely 
re-written  the  account  of  the  bichromate  of  silver  impregna- 
tions of  Golgi  ;  the  account  as  it  now  stands  is,  I  believe, 
the  only  complete  one  that  has  appeared  in  the  English 
language. 

I  am  under  the  greatest  obligation  to  Professor  van 
Gehuchten,  as  well  as  to  Dr.  Paul  Mayer,  for  the  generous 
assistance  which  enables  me  to  affirm  that  the  important 
subjects  in  question  have  been  treated  with  all  the  requisite 
accuracy  and  thoroughness. 

The  essential  feature  of  the  first  edition  was  that  it  was  an 
altogether  exhaustive  collection  of  all  the  methods  of  pre- 
paration that  had  up  to  that  time  been  recommended  as 
useful  for  the  purposes  of  microscopic  anatomy,  and  its 
primary  intention  that  of  being  a  work  of  reference  for  the 
instructed  anatomist.  Its  character  of  a  guide  to  the  be- 
ginner was  secondary  only.  It  contained,  indeed,  a  general 
introduction  and  much  explanatory  matter  in  the  different 
chapters,  but,  on  the  whole,  the  didactic  matter  bore  but  an 
insufficient  proportion  to  the  historical  matter.  This  has  now 
been  rectified.  It  has  come  to  pass  that  during  the  repeated 
operations  of  revision  to  which  the  book  has  been  subjected, 
the  explanatory  and  didactic  element  has  been  continually 
increasing,  whilst  at  the  same  time  the  historical  element 
Iia-  been  continually  diminishing — diminishing,  that  is,  in  all 
parts  of  the  book  relatively  to  the  former  element,  and  in 
some  parts  absolutely  (as  may  be  seen,  for  instance,  by  com- 
paring the  number  of  formulae  given  in  the  chapters  on 
Carmine  and  Haematoxylin  with  the  number  given  in  former 
editions).  On  the  one  hand  the  book  has  been  lightened  by 


X  PEEFACE. 

the  jettison  of  much  useless  matter,  and  on  the  other  hand 
there  has  been  accorded  to  the  matter  that  has  been  retained 
a  far  ampler  share  than  before  of  explanation  and  detail. 
To  such  an  extent,  indeed,  nave  the  instructions  to  students 
and  other  explanatory  matter  been  amplified  that  I  am  not 
acquainted  with  any  modern  work  on  the  subject  that  con- 
tains anything  like  so  complete  an  account  of  the  various 
fundamental  operations  of  histological  technique — fixing,  im- 
bedding, staining,  and  the  like.  I  only  felt  justified  in 
claiming  for  the  first  edition  that  it  "  went  far  to  make  up 
a  formal  treatise  on  the  art."  Through  the  changes  above 
mentioned  the  book  has  come  to  assume  altogether  the 
character  of  a  formal  treatise,  and  now  contains  in  due  pro- 
portions both  the  grammar  and  the  dictionary  of  the  firt. 

The  rejection  of  superfluous  matter  above  referred  to  re- 
lates chiefly  to  old  methods  that  have  been  before  the  public 
for  so  long  a  time  that  there  can  be  no  doubt  that  they 
have  no  good  claim  to  further  survival,  whilst  recent  methods, 
which  may  be  considered  to  be  still  on  their  probation,  have 
been  treated  with  the  accustomed  fulness*. 


NYON,  SWITZERLAND  ; 

September,  1896. 


CONTENTS. 

PART   I. 


PAGE 

CHAPTER  I. 


INTRODUCTORY  . 


CHAPTER   II. 
KILLING  ......  .11 

CHAPTER   III. 
FIXING  AND  HARDENING          .  .  .  .  .  .19 

CHAPTER   IV. 

FIXING    AND    HARDENING    AGENTS;    MINERAL    ACIDS   AND   THEIR 

SALTS          ........      31 

CHAPTER  V. 

FIXING   AND    HARDENING   AGENTS;    CHLORIDES,  ORGANIC   ACIDS, 

AND  OTHERS  .  .  .  .  .  .  .54 

CHAPTER  VI. 
DE-ALCOHOLISATION  AND  CLEARING  AGENTS  .  .  .  .78 

CHAPTER  VII. 
IMBEDDING  METHODS — INTRODUCTION  .  .  .  .87 

CHAPTER  VIII. 

IMBEDDING  METHODS:  PARAFFIN  AND  OTHER  FUSION  MASSES        .      96 
Paraffin,  96;  Gelatin,  117. 


xii  CONTENTS. 

PAGE 

CHAPTER  IX. 

COLLODION  (CELLOIDIN)  AND  OTHEE  IMBEDDING  METHODS  .  .    120 

Collodion  or  Celloidin,  320;  other  Cold  Masses,  135;  Freezing, 
137. 

CHAPTER   X. 

SERIAL  SECTION  MOUNTING     . 

Methods  for  Paraffin  Sections,  140  ;  Methods  for  Watery  Sections, 
146  ;  Methods  for  Celloidin  Sections,  147. 

CHAPTER  XI. 

STAINING  .  '    153 

CHAPTER  XII. 

CARMINE  AND  COCHINEAL  STAINS 

Theory  of  Carmine  Staining,  163  ;  Aqueous  Carmines,  Acid,  167  ; 
Neutral  and  Alkaline,  171  ;  Alcoholic  Carmines  and  Cochineals, 
172. 

CHAPTER  XIII. 

HJEMATEIN  (HJSMATOXYLIN)  STAINS    . 

Theory  of  Staining  with  Hsematoxylin,  177  ;  Alumina-hsematein 
Lakes,  182  ;  other  Hsematein  Compounds,  187. 

CHAPTER  XIV. 

ON  STAINING  WITH  COAL-TAR  COLOURS 

Basic,  Acid  and  Neutral  Coal-tar  Colours,  193  ;  Progressive  and 
Regressive  Stains,  195 ;  General  Directions  for  the  Regressive 
Method,  196. 

CHAPTER  XV. 

THE  COAL-TAR  CHROMATIN  STAINS      . 

Regressive  Stains,  202  ;  Progressive  Stains,  208. 

CHAPTER   XVI. 
THE  COAL-TAR  PLASMA  STAINS 

CHAPTER  XVII. 
METHYLEN  BLUE  ...  •  228 


CONTENTS.  Xlii 

PAGE 

CHAPTER  XVIII. 

METALLIC  STAINS  (IMPREGNATION  METHODS)  .  .  .    241 

Silver,  243  ;  Gold,  249  ;  other  Metallic  Stains,  258. 

CHAPTER  XIX. 

OTHER  STAINS  AND  COMBINATIONS      .....    262 
Other  Organic  Stains,  262  ;  Carmine  combinations,  264 ;  Haema- 
te'in  Combinations,  266. 

CHAPTER  XX. 

EXAMINATION  AND  PRESERVATION  MEDIA      ....    268 
Aqueous  Liquids,  268  ;    Mercurial  Liquids,   272  ;  other  Fluids, 
273 ;  Glycerin  Media,  276 ;  Jellies,  278  ;  Resinous  Media,  281. 

CHAPTER  XXI. 

CEMENTS  AND  VARNISHES  288 


PART    II. 
SPECIAL   METHODS   AND   EXAMPLES. 


CHAPTER  XXII. 

INJECTIONS  :  GELATIN  MASSES  .....    297 

Carmine,  300  ;  Blue,  303  ;  other  Colours,  305. 

CHAPTER  XXIII. 

INJECTIONS:   OTHER  MASSES  (COLD)    .....    307 
White  of  Egg,  Gum,  307;  Glycerin,  308;  Aqueous,  309;   Cel- 
loidin,  310 ;  other  Masses,  310. 

CHAPTER  XXIV. 

MACERATION  AND  DIGESTION  ......    312 

Maceration,  312  ;  Digestion,  319. 

CHAPTER  XXV. 

CORROSION,  DECALCIFICATION,  DESILICIFICATION,  AND  BLEACHING    .     321 
Corrosion,  321;  Decalcification,  322;  Desilicification,  326;  Bleach- 
ing, 327, 

c 


XIV  CONTESTS. 


PAGE 

CHAPTEE  XXVI. 
EMBRYOLOGICAL  METHODS  331 


CHAPTER  XXVII. 
CYTOLOGICAL  METHODS  ......     357 

CHAPTER  XXVIII. 
TEGUMENTARY  ORGANS  ......     366 

CHAPTER  XXIX. 

MUSCLE  AND  TENDON  (NERVE-ENDINGS)         ....     371 
Striated  Muscle,  371 ;  Tendon,  373  ;  Smooth  Muscle,  374. 

CHAPTER  XXX. 

NEUROLOGICAL  METHODS. — INTRODUCTION  AND  SECTION  METHODS    .    377 

CHAPTER  XXXI. 

NEUROLOGICAL     METHODS. — NERVE-FIBRE    STAINS    (Weigert    and 

others)         .........     396 

CHAPTER  XXXII. 

NEUROLOGICAL  METHODS. — AXIS-CYLINDER  AND  PROTOPLASM  STAINS 

(Golgi  and  others)  .......     409 

Neuroglia,  428;  Retina,  431 ;  Inner  ear,  433. 

CHAPTER   XXXIII. 

SOME    OTHER    HlSTOLOGICAL    METHODS  ....      435 

Connective  Tissues,  435;  Blood,  447  ;  Glands,  453. 

CHAPTER   XXXIV. 

SOME  METHODS  FOR  LOWER  ANIMALS  ....     459 

Tunicata,  459  ;  Mollusca,  461  ;  Arthropoda,  465  ;  Vermes,  469 ; 
Echinodermata,  480;  Ccelenterata,  484;  Porifera,  489;  Pro- 
tozoa, 490. 

APPENDIX          ........    497 

INDEX  503 


THE  MICROTOMISrS  VADE-MECUM. 


CHAPTER  I. 
INTRODUCTORY. 

1.  The  General  Method. — The  methods  of  modern  micro- 
scopic anatomy  may  be  roughly  classed  as  General  and 
Special.  There  is  a  General  or  Normal  method,  known  as 
the  method  of  sections,  which  consists  in  carefully  fixing  the 
structures  to  be  examined,  staining  them  .with  a  nuclear 
stain,  dehydrating  with  alcohol,  and  mounting  series  of  section* 
of  the  structures  in  balsam.  It  is  by  this  method  that  the 
work  is  blocked  out  and  very  often  finished.  Special  points 
are  then  studied,  if  necessary,  by  Special  Methods,  such  as 
examination  of  the  living  tissue  elements,  in  situ  or  in 
"  indifferent  "  media  ;  fixation  with  .  special  fixing  agents  ; 
staining  with  ^special  stains;  dissociation  by  teasing  or 
maceration  ;  injection  ;  impregnation  ;  and  the  like. 

There  is  a  further  distinction  which  may  be  made,  and 
which  may  help  to  simplify  matters.  The  processes  of  the 
preparation  of  tissues  may  be  divided  into  two  stages,  Pre- 
liminary Preparation  and  Ulterior  Preparation.  .  Now  the 
processes  of  preliminary  preparation  are  essentially  identical 
in  all  the  methods,  essential  divergences  being  only  found 
in  the  details  of  ulterior  preparation.  By  preliminary  pre- 
paration is  meant  that  group  of  processes  called  by  German 
anatomists  Ccmseruirungsmethoden, — those,  namely,  whose 
object  it  is  to  get  the  tissues  into  a  fit  state  for  passing  un- 
harmed through  all  the  ulterior  processes  to  which  it  may 
be  desired  to  submit  them.  Preliminary  preparation  com- 
prehends the  operations  of  (1)  killing;  (2)  fixing;  (3)  the 
washing  and  other  manipulations  necessary  for  removing  the 


2  CHAPTER   I. 

fixing  agent  from  the  tissues,  and  substituting  for  it  the 
preservative  liquid  or  other  reagents  which  it  is  desired  to 
employ.  Ulterior  preparation  comprehends  the  processes 
sketched  out  in  §§  3  et  seq. 

2.  Preliminary  Preparation. — The  first  thing  to  be  done 
with  any  structure  is  to  fix  its  histological  elements.  (This 
statement  applies  equally  to  all  classes  of  objects,  whether 
it  be  desired  to  cut  them  into  sections  or  to  treat  them  in 
any  other  special  way.)  Two  things  are  implied  by  the 
word  "  fixing  :  "  first,  the  rapid  Jailing  of  the  element,  so 
that  it  may  not  have  time  to  change  the  form  it  had  during 
life,  but  is  fixed  in  death  in  the  attitude  it  normally  had 
during  life  ;  and  second,  the  hardening  of  it  to  such  a  degree 
as  may  enable  it  to  resist  without  further  change  of  form  the 
action  of  the  reagents  with  which  it  may  subsequently  be 
treated.  Too  much  stress  can  hardly  be  laid  on  this  point, 
which  is  the  most  distinctive  feature  of  modern  histological 
practice  ;  without  good  fixation  it  is  impossible  to  get  good 
stains  or  good  sections,  or  preparations  good  in  any  way. 

The  structure  having  been  duly  fixed  by  one  of  the  pro- 
cesses described  in  the  chapter  on  Fixing  Agents,  is  washed 
in  order  to  remove  from  the  tissues  as  far  as  possible  all 
traces  of  the  fixing  reagent. 

The  kind  of  liquid  witli  which  washing  out  is  done  is  not  a  matter  of  in- 
difference. If  corrosive  sublimate  (for  instance),  or  osmic  acid,  or  a  solution 
into  which  chromic  acid  or  a  chromate  enters,  have  been  used  for  fixing,  the 
washing  may  be  done  with  water.  But  if  picric  acid  in  any  form,  has  been 
used,  the  washing  must  be  done  with  alcohol.  The  reason  of  this  difference 
is  that  the  first-named  reagents  (and,  indeed,  all  the  compounds  of  the 
heavy  metals  used  for  fixing)  enter  into  a  state  of  chemical  combination 
with  the  elements  of  tissues,  rendering  them  insoluble  in  water;  so  that  the 
hardening  induced  by  these  agents  is  not  rerqoved  by  subsequent  treatment 
with  water.  Picric  acid,  on  the  other  hand,  produces  only  a  very  slight 
hardening  of  the  tissues,  so  that  the  tissue  elements  are  left  in  a  soft 
state  in  which  they  are  obnoxious  to  all  the  hurtful  effects  of  water. 
Alcohol  must  therefore  be  taken  to  remove  the  picric  acid  and  to  effect  the 
necessary  hardening  at  the  same  time.  Instructions  for  washing  out  are 
given,  when  necessary,  in  the  discussion  of  the  different  fixing  agents  in  the 
following  parts  of  this  work. 

These  operations  having  been  duly  performed,  two  roads 
become  open.  The  object  may  be  further  prepared  by  what 


INTRODUCTORY.  3 

may  be  termed  the  ^wet  method,  in  which  all  subsequent 
operations  are  performed  by  means  of  aqueous  media.  Or  it 
may  be  further  prepared  by  what  may  be  termed  the  Dehy- 
dration method,  which  consists  in  treatment  with  successive 
alcohols  of  gradually  increasing  strength,  final  dehydration 
with  absolute  alcohol,  imbibition  with  an  essential  oil  or 
other  so-called  clear  liuj  agtnt  which  serves  to  remove  the 
alcohol,  and  lastly  either  mounting  at  once  in  balsam  or 
other  resinous  medium  or  imbedding  in  paraffin  for  the 
purpose  of  making  sections.  The  dehydration  method  is  the 
course  which  is  generally  preferred,  chiefly  because  of  its 
great  superiority  as  regards  the  jDreservation  of  tissues.  The 
presence  of  water  is  the  mo'st  important  factor  in  the  con- 
ditions that  bring  about  the  decomposition  of  organic  matter, 
and  its  complete  removal  is  the  chief  condition  of  permanent 
preservation.  It  is  of  course  not  intended  here  to  suggest 
that  wet  methods  of  preparation  should  be  altogether  dis- 
carded. They  have  great  value,  they  are  even  indispensable 
for  special  ends  ;  and  all  that  is  intended  to  be  suggested  is 
that  they  should  be  regarded  not  as  ^general,  but  as,  special 
methods. 

3.  Dehydration  and  Preservation. — The  further  course  of 
preparation  by  the  dehydration  method  is  as  follows  : — At 
the  same  time  that  the  superfluous  fixing  agent  is  being  re- 
moved from  the  tissues,  or  as  soon  as  that  is  done,  the  water 
of  tlte  tisane*  'must  be  removed.  This  is  necessary  for  two 
reasons  :  firstly,  in  the  interest  of  preservation,  the  presence 
of  .water  being  the  condition  of  all  others  that  most  favours 
post-mortem  decomposition ;  and  secondly,  because  all  water 
must  be  removed  in  order  to  allow  the  tissues  to  be  im- 
pregnated with  the  imbedding  material  necessary  for  section - 
cutting,  or  with  the  balsam  with  which  they  are  to  be  finally 
preserved.  (The  cases  in  which  aqueous  imbedding  and 
preserving  media  are  employed  are  exceptional,  and  will  be 
treated  of  in  the  proper  places.)  This  dehydration  is  per- 
formed as  follows  : — The  objects  are  brought  into  weak 
alcohol,  and  are  then  passed  through  successive  alcohols  of 
gradually  increased  strength,  remaining  in  each  the  time 
necessary  for  complete  saturation,  and  the  last  bath  consist- 
ing of  absolute  or  at  least  very  strong  alcohol. 


4  CHAPTER  I. 

In  dealing  with^extremely  delicate  objects,  it  may  be  necessary  to  take 
special  precautions  in  order  to  avoid  injury  to  them  through  the  violent 
diffusion-currents  that  are  set  up  in  the  passage  from  water  to  alcohol,  or 
from  one  bath  of  alcohol  to  another  of  considerably  different  density.  Some 
kind  of  diffusion-apparatus  may  conveniently  be  used  in  these  cases.  The 
objects  may  be  placed  with  some  of  their  liquid  in  a  tube  plugged  at  one 
end  and  closed  at  the  other  by  a  diaphragm  of  chamois  skin  or  other  suitable 
membrane,  the  tube  being  then  immersed  in  a  vessel  containing  the  grade 
of  alcohol  that  it  is  desired  to  add  to  the  liquid  in  the  tube,  and  the  whole 
allowed  to  remain  until  by  diffusion  through  the  diaphragm  the  two  liquids 
have  become  of  equal  density.  Or  COBB'S  differentiator  (Proc.  Linn.  Soc., 
N.S.W.,  v,  1890,  p.  157 ;  Journ.  Roy.  Mic.  Soc.,  1890,  p.  821)  may  be 
employed.  Or,  more  conveniently  in  most  cases,  the  apparatus  described 
and  figured  by  HASWELL  (Proc.  Linn.  Soc.,  N.S.W.,  vi,  1891,  p.  433  ; 
Journ.  Roy.  Mic.  Soc.,  1892,  p.  696).  This  consists  of  two  wash-bottles 
connected  in  the  usual  way  by  tubing,  and  furnished,  the  one  with  an  over- 
flow-tube, and  the  other  with  a  feeding-tube  leading  from  an  elevated 
reservoir  connected  with  it  by  means  of  a  regulating  tap  or  drop  arrange- 
ment. The  objects  are  placed  in  the  first  bottle ;  some  of  the  same  liquid 
as  that  containing  the  objects  is  placed  in  the  second  bottle ;  and  alcohol  of 
the  grade  that  it  is  desired  to  add  is  led  into  it  from  the  reservoir.  The 
mixture  of  liquids  therefore  takes  place  in  the  bottle  that  does  not  contain 
the  objects,  and  the  mixture  itself  is  gradually  led  over  to  the  objects 
through  the  siphon-tube  connecting  the  two  bottles.  Another  apparatus 
for  rapid  dehydration,  devised  by  CHEATLE,  will  be  found  described  in 
Journ.  Pathol.  and  Bacteriol.,  i,  1892,  p.  253,  or  Journ.  Roy.  Mic.  Soc., 
1892,  p.  892.  It  is  hardly  simple  enough  to  be  recommendable. 

A  capillary  siphon  for  the  aspiration  of  liquids  in  the 
fixing,  staining,  and  washing  of  suspended  blood-corpuscles, 
sperm-cells,  protozoa,  and  the  like,  is  described  by  EWALD, 
in  Zeit.  BioL,  Bd.  xxxiv,  1897,  p.  253. 

I  would  here  call  attention  to  the  varied  usefulness  of  the  "  Siebdosen  " 
or  sieve-dishes  of  STEINACH,  ZIMMEEMANN,  and  SUCHANNEK  (vide  Zeit.  f. 
wiss.  Mik.,  iv,  4,  1887,  p.  433,  and  vii,  2,  1890,  p.  158).  They  consist  of  a 
covered  glass  capsule  into  which  is  fitted  a  "  sieve  "  made  of  a  watch-glass 
pierced  with  holes  and  supported  on  legs,  and  are  very  handy,  not  only  for 
staining,  washing  out,  treatment  with  vapours,  etc.,  but  for  any  operation 
in  which  it  is  desirable  to  have  specimens  supported  in  the  upper  layers  of 
a  quantity  of  reagent.  They  are  sent  out  in  a  very  neat  form  by  Griibler 
and  Co.  FAIECHILD'S  perforated  porcelain  cylinders  for  washing  (Zeit.  f. 
wiss.  Mik.,  xii,  3,  1896,  p.  301)  seem  to  be  a  very  neat  idea.  These  are 
made  small  enough  to  be  floated  by  the  cork  that  closes  them.  See  also 
EWALD'S  section-washing  apparatus  (Zeit.  BioL,  Bd.  xxxiv,  1897,  p.  264). 

It  is  sometimes  stated  that  it  is  necessary  that  the  last  alcohol-bath 
should  consist  of  absolute  alcohol.  This,  however,  is  incorrect,  a  strength 
of  90  per  cent.,  or  at  all  events  95  per  cent.,  being  sufficient  in  most  cases. 
For  the  small  amount  of  water  that  remains  in  the  tissues  after  treatment 
with  these  grades  of  alcohol  is  efficiently  removed  in  the  bath  of  clearing 


INTRODUCTORY.  5 

agent  if  a  good  clearing  agent  be  employed.  Oil  of  cedar  will  remove  the 
remaining  water  from  tissues  saturated  with  95  per  cent,  alcohol ;  oil  of 
bergamot  will  **  clear  "  from  90  per  cent,  alcohol,  and  anilin  oil  will  clear 
from  70  per  cent,  alcohol. 

I  am  not  aware  of  any  substance  that  can  entirely  take  the  place  of 
alcohol  for  dehydration  and  preservation.  Acetone  and  inethylal  have  been 
substituted  for  alcohol  in  the  dehydration  of  methylen-blue  preparations 
(PARKER,  ZooL  Anz.,  403,  1892,  p.  376) ;  but  a  really  efficient  substitute 
for  alcohol  in  general  work  remains  yet  to  be  discovered.  Formaldehyde 
(see  under  Fixing  and  Hardening  Agents)  is  now  known  to  be  a  most 
admirable  medium  for  the  preservation  of  museum  specimens,  being  for  that 
purpose  in  many  cases  greatly  superior  to  alcohol ;  but  experience  is  want- 
ing as  to  how  far  it  is  available  for  the  preservation  of  histological  material, 
whilst  of  course,  occurring  as  it  does  in  the  form  of  an  aqueous  solution,  it 
can  have  no  dehydrating  effect. 

Considered  as  a  mere  dehydrating  agent,  alcohol  fulfils 
its  functions  fairly  well.  But  considered  as  a  histological 
preservative  agent,  it  is  far  less  satisfactory.  If  tissues  be 
left  in  alcohol  for  only  a  few  days  before  further  prepara- 
tion, the  injurious  effects  of  a  sojourn  in  alcohol  will  perhaps 
not  be  very  disagreeably  evident.  But  it  is  otherwise  if 
they  are  put  away  in  it  for  many  weeks  or  months  before 
the  final  preparation  is  carried  out.  The  dehydrating  action 
of  the  alcohol  being  continuously  prolonged,  the  minute 
structure  of  tissues  is  sometimes  considerably  altered  by  it; 
they  become  ,over-hard  and  shrink,  and  become  brittle,  and 
their  ^capacity  for  taking  stains  well  becomes  seriously 
diminished.  KDLTSCHITZKY  (Zeit.  f.  rviss.  Mik.,  iv,  3,  1887, 
p.  349)  has  proposed  to  remed}7  this  by  putting  up  objects, 
after  fixation  and  washing  out  with  alcohol,  in  ether,  xylol, 
or  toluol.  FI.EMMING  (Arch.  f.  milt.  Anat.,  xxxvii,  1891, 
p.  685)  advises  putting  up  objects  after  fixation  in  a  mixture 
of  alcohol,  glycerin,  and  water,  in  about  equal  parts,  pointing 
out  that  objects  thus  preserved  may  be  at  any  *  moment 
either  prepared  for  sectioning  by  treatment  with  pure  alcohol 
or  softened  for  dissection  or  teasing  by  a  little  soaking  in 
water,  and  that  they  do  not  become  so  hard  and  brittle  as 
alcohol  specimens,  and  retain  their  stnining  power  much 
better.  After  extensive  experience  of  this  plan  I  can  highly 
recommend  it,  and  would  only  further  suggest  that  the  action 
of  the  liquid  seems  to  me  to  be  in  many  cases  much  im- 
proved by  addition  of  a  little  acetic  acid  (say  0'5  to  0*75 
per  cent.). 


6  CHAPTER   I. 

For  material  that  is  intended  only  for  section- cutting,  1 
find  that  by  far  the  best  plan  is  to  clear  and  imbed  at  once 
in  paraffin.  This  affords,  as  far  as  I  can  see,  an  absolutely 
perfect  preservation.  I  have  worked  on  material  that  has 
been  preserved  in  this  way  for  over  seven  years.  The  pre- 
servation of  the  tissues,  down  to  the  finest  details  of  cell- 
structure,  appears  to  be  perfect,  and  the  staining  as  precise 
as  when  the  specimens  were  first  put  up.  The  only  notice- 
able defect  is  that  the  tissues  are  rather  brittle,  and  do  not 
Gut  well ;  but  it  is  not  certain  that  that  is  not  owing  to  their 
having  been  over-hardened  in  the  first  instance.  Cedar- wood 
oil  is,  I  find,  nearly,  if  not  quite,  as  good  as  paraffin. 

4.  Removal  of  Alcohol ;  Clearing, — The  water  having  been 
thus  sufficiently  removed,  the  alcohol  is  in  its  turn  removed 
from  the  tissues,  and  its  place  taken  by  some  anhydrous 
substance,  generally  an  essential  oil,  which  is  miscible  with 
the  material  used  for  imbedding  or  mounting.  This  opera- 
tion is  generally  known  as  Clearing.  It  is  very  important 
that  the  passage  from  the  last  alcohol  to  the  clearing  agent 
be  made  gradual.  This  is  effected  by  placing  the  clearing 
medium  under  the  alcohol.  A  sufficient  quantity  of  alcohol 
is  placed  in  a  tube  (a  watch-glass  will  do,  but  tubes  are 
generally  better),  and  then  with  a  pipette  a  sufficient  quantity 
of  clearing  medium  is  introduced  at  the  bottom  of  the  alcohol. 
Or  you  may  first  put  the  clearing  medium  into  the  tube,  and 
then  carefully  pour  the  alcohol  on  to  the  top  of  it.  The  two 
fluids  mingle  but  slowly.  The  objects  to  be  cleared,  being 
now  quietly  put  into  the  supernatant  alcohol,  float  at  the 
surface  of  separation  of  the  two  fluids,  the  exchange  of 
fluids  takes  place  gradually,  and  the  objects  slowly  sink 
down  into  the  lower  layer.  When  they  have  sunk  to  the 
bottom,  the  alcohol  may  be  drawn  off  with  a  pipette,  and 
after  some  further  lapse  of  time  the  objects  will  be  found  to 
be  completely  penetrated  by  the  clearing  medium.  This 
method  of  making  the  passage  from  one  fluid  to  another 
applies  to  all  cases  in  which  objects  have  to  be  transferred 
from  a  lighter  to  a  denser  fluid, — for  instance,  from  alcohol, 
or  from  water,  to  glycerin. 

It  should  be  noted  here  that  this  is  the  proper  stage  for 
carrying  out  minute  dissections,  if  any  such  have  to  be  done, 


INTRODUCTORY.  7 

a  drop  of  clearing  agent  being  a  most  helpful  medium  for 
carrying  out  such  dissections  (see  §  8). 

At  this  point  the  course  of  treatment  follows  one  of  two 
different  roads,  according  as  the  object  is  to  be  mounted 
direct  in  balsam  (§  7),  or  is  first  to  be  sectioned  (§5). 

5.  Imbedding,  and  Treatment  of  Sections. — The  objects  are 
now  imbedded.  They  are  removed  from  the  clearing  medium, 
and  soaked  until  thoroughly  saturated  in  the  imbedding 
medium.  This  is,  for  small  objects,  generally  paraffin, 
liquefied  by  heat,  and  for  large  objects  generally  a  solution 
of  collodion  or  "  celloidin  "  (in  this  latter  case  the  clearing 
may  be  omitted  and  the  tissues  be  imbedded  direct  from  the 
alcohol).  The  imbedding  medium  containing  the  object  is 
then  made  to  solidify,  as  described  in  the  chapter  on  Im- 
bedding Processes,  and  sections  are  made  with  a  microtome 
through  the  imbedding  mass  and  the  included  objects.  The 
sections  are  then  mounted  on  a  slide  by  one  of  the  methods 
described  in  the  chapter  on  Serial  Section  Methods,  the 
imbedding  material  is  removed  from  them  (in  the  case  of 
paraffin),  they  are  stained  in  situ  on  the  slide,  dehydrated 
with  alcohol,  cleared,  and  mounted  in  balsam  or  damar.  Or 
they  may  be  stained,  washed,  dehydrated,  and  cleared  in 
watch-glasses,  and  afterwards  mounted  as  desired — the  im- 
bedding medium  being  first  removed  if  desirable. 

The  plan  of  staining  sections  on  the  slide  is  of  somewhat  recent  introduc- 
tion ;  before  it  had  been  worked  out  the  practice  was  to  stain  structures  in 
toto,  before  cutting  sections.  In  this  case  the  object,  after  having  been 
fixed  and  washed  out,  is  taken  from  the  water,  or  while  still  on  its  way 
through  the  lower  alcohols  (it  should  not  be  allowed  to  proceed  to  the 
higher  grades  of  alcohol  before  staining,  if  that  can  be  avoided),  and  passed 
through  a  bath  of  stain  (generally  alcoholic  borax-carmine  or  other  alcoholic 
stain)  of  sufficient  duration,  then  dehydrated  with  successive  alcohols,  passed 
through  a  clearing  medium  into  paraffin,  cut,  and  treated  as  above  described, 
the  sections  in  this  case  being  mounted  direct  from  the  chloroform,  xylol, 
or  other  solvent  with  which  the  paraffin  is  removed.  If  aqueous  staining 
media  be  applied  (and  this  is  sometimes  very  desirable  for  particular  pur- 
poses) the  structures  should  either  be  stained  in  toto  immediately  after 
fixing  and  washing  out,  or  sections  may  be  stained  on  the  slide,  the  objects, 
if  delicate,  being  passed  through  successive  baths  of  alcohol  of  gradually 
decreasing  strength  before  being  put  into  the  aqueous  stain. 

In  my  opinion  it  is  generally  advisable  not  to  stain  in  bulk  material  that 
is  intended  to  be  sectioned;  by  staining  it  as  sections  the  staining  can  be 


8  .CHAPTER    I. 

much  better  controlled,  and  many  excellent  stains  can  in  this  way  be  em- 
ployed that  are  not  available  for  staining  in  bulk  ;  and  of  course  sections 
can  be  stained  much  more  rapidly  than  material  in  bulk.  But  many 
workers  consider  that  staining  in  bulk  is  frequently  more  convenient,  and 
therefore  preferable  so  long  as  the  demonstration  of  minute  detail  is  not  an 
object. 

The  most  convenient  vessels,  I  find,  in  which  to  perform  the  various 
operations  of  staining,  differentiating,  dehydrating,  clearing,  etc.,  on  the 
slide,  are  flat-bottomed  corked  glass  tubes.  I  have  mine  made  10  centi- 
metres high  and  27  millimetres  internal  diameter.  Each  of  these  will  then 
take  two  slides,  English  size,  placed  back  to  back.  To  make  a  stand  for 
them,  take  a  piece  of  deal  board,  3  centimetres  thick,  and  with  a  centrebit 
bore  in  it  series  of  holes  about  15  millimetres  deep  and  of  the  diameter  of 
the  tubes,  and  about  3  centimetres  apart  lengthways  and  1|  crossways.  A 
board  of  15  centimetres  width  and  45  length  will  take  twenty-one  tubes  in 
three  rows  of  seven  each  in  the  holes  ;  and  others  may  be  stood  up  between 
the  rows  without  much  risk  of  their  falling. 

6.  Resume  of  the  Section  Method. — It  was  stated  in  the  first 
edition  of  tlds  work  that  l<  the  great  majority  of  preparations 
are  made  by  fixing  either  with  sublimate  or  a  picric  acid  com- 
bination, washing  out  with   alcohol,  staining  with  alcoholic 
borax-carmine,    imbedding    in    chloroform    paraffin,    cutting 
with  a  sliding  microtome,  and  mounting  the  sections  in  series 
in  Canada  balsam."      But  histological  practice  has  greatly 
changed  since  then,  and  I  would  now  suggest  the  following 
as  giving  in  very  many  cases  greatly  superior  results  : — Fix 
in    such    one    of  the   fixing    agents    recommended    in    later 
chapters  as  may  be  most   suitable    to  the   case ;   wash   out ; 
dehydrate  ;  clear  with  oil  of  cedar- wood  ;  imbed  in  paraffin  ; 
mount   sections  on  the  slide   by  the  water   method  or  with 
Mayer's  albumen  medium;   stain  as  desired,  and  mount    in 
balsam  or  damar.      That,  or  something  like  that,  is  now  the 
practice   of   many  of    the  most  advanced  workers ;   but  the 
beginner  will  perhaps  do  well  to   commence  by  the   simpler 
procedure   first    recommended,   which    is   very    suitable   for 
obtaining  rapidly  a  general  view  of  the  forms  and  relations 
of  anatomical  elements. 

7.  Preparation  of  Entire  Objects,  or  of  Material  that  is  not  to 
be  sectioned. — The  treatment  of  objects  which  can  be  studied 
without  being  cut  into  sections  is  identical  with  that  above 
described,  with  the  omission  of  those  passages  that  relate  to 
imbedding  processes.      Its  normal  course  may  be  described 


IXI'UODTJCTOUY.  9 

as  fixation,  washing  out,  staining,  treatment  with  successive 
alcohols  of  gradually  increasing  strength,  final  dehydration 
with  absolute  alcohol,  clearing,  and  mounting  in  balsam. 
This  method  is  usually  preferred,  as  a  general  method,  to  the 
\vi't  methods,  for  the  reasons  that  have  been  given  above 
(§  2),  and  for  some  others,  amongst  which  may  be  noted  the 
greater  transparency  given  to  tissues  by  mounting  them  in 
media  of  high  refractive  index,  such  as  balsam. 

In  the  preparation  of  entire  objects  or  structures  that  are 
intact  and  covered  by  an  integument  not  easily  permeable  by 
liquids,  special  care  must  be  taken  to  avoid  swelling  from 
endosmosis  on  the  passage  of  the  objects  from  any  of  the 
liquids  employed  to  a  liquid  of  less  density,  or  shrinkage  from 
exosmosis  on  the  passage  to  a  liquid  of  greater  density.  This 
applies  most  specially  to  the  passage  from  the  last  alcohol 
into  the  clearing  medium.  A  slit  should  be  made  in  the 
integument,  if  possible,  so  that  the  two  fluids  may  mingle 
without  hindrance.  And  in  all  cases  the  passage  is  made 
gradual  by  placing  the  clearing  medium  under  the  alcohol, 
as  above  described.  Fluids  of  high  di (fusibility  should  be 
employed  as  far  as  possible  in  all  the  processes.  Fixing 
agents  of  great  penetrating  power  (such  as  picro-sulphuric 
acid  or  alcoholic  sublimate  solution)  should  be  employed 
where  the  objects  present  a  not  easily  permeable  integument. 
Washing  out  is  done  with  successive  alcohols,  w^ater  being 
used  only  in  the  case  of  fixation  by  osmic  acid,  or  the  chromic 
mixtures  or  other  fixing  solutions  that  render  washing  by 
water  imperative.  Staining  is  done  by  preference  with 
alcoholic  staining  media.  The  stains  most  to  be  recommended 
are  Grenadier's  borax-carmine,  or  one  of  Mayer's  new 
carminic  acid  or  haematein  stains  (for  all  of  which  see 
STAINING  AGKNTS).  Tar-colour  stains  are  rarely  applicable  to 
this  class  of  preparations.  Aqueous  stains  are  more  rarely 
indicated,  though  there  are  many  cases  in  which  they  are 
admissible,  and  some  in  which  they  are  preferable. 

8.  Minute  Dissections. — These  are  best  done,  if  necessary, 
in  a  drop  of  clearing  agent.  I  recommend  cedar-wood  oil 
for  this  purpose,  as  it  gives  to  the  tissues  a  consistency  very 
favourable  for  dissection,  whilst  its  viscosity  serves  to  lend 
support  to  delicate  structures.  Clove  oil  lias  a  tendency  to 


10  CHAPTEE    I. 

make  tissues  that  have  lain  in  it  for  some  time  very  brittle. 
The  brittleness  is,  however,  sometimes  very  helpful  in  minute 
dissections.  Another  property  of  clove  oil  is  that  it  does  not 
easily  spread  itself  over  the  surface  of  a  slide,  but  has  a 
tendency  to  form  very  convex  drops,  and  this  also  makes  it 
frequently  a  very  convenient  medium  for  making  minute 
dissections  in. 

9.  General  Principles. — For  an  excellent  exposition  of  the  principles 
underlying  modern  histological  technique,  the  reader  may  consult  with 
advantage  the  paper  of  PAUL  MAYER,  in  Mitth.  ZooL  Stat.  Neapel,  ii 
(1881),  p.  1,  et,  seq.  See  also  the  abstract  in  Journ.  Roy.  Mic.  boc.  (N.S.), 

11  (1882),  pp.  866—881,  and  that  in  Amer.  Natural,  xvi  (1882),  pp.  697— 
706,  in  which  two  last  some  improvements  are  mentioned  which  have  been 
worked  out  since  the  publication  of  Mayer's  paper ;  and  further,  the  history 
and  criticism  of  modern  methods  contained  in  APATHY'S  Mikrotechnik  der 
thierischen  Morphologic,  Braunschweig,  H.  Bruhn,  1896. 


CHAPTER   II. 

KILLING. 

10.  IN  the  majority  of  cases,  the  first  step  in  the  prepara- 
tion   of   an   organ    or   organism   consists    in    exposing  it    as 
rapidly  and  as  completely  as  possible  to  the  action  of  one  of 
the  Fixing  Agents  that   are   discussed   in  the   next  chapter. 
The  organ  or  organism  is  thus  taken  in  the  normal  living 
state  ;   the  fixing  agent  serves  to  bring  about  at  the  same 
time,    and   with  sufficient   rapidity,   both    the   death  of   the 
organism  and  that  of  its  histological  elements. 

But  this  method  is  by  no  means  applicable  to  all  cases. 
There  are  many  animals,  especially  such  as  are  of  a  soft  con- 
sistence, and  deprived  of  any  rigid  skeleton,  but  possessing 
a  considerable  faculty  of  contractility —  such  as  many  Ccelen- 
terata,  Bryozoa,  and  Serpulida,  for  instance — which  if  thus 
treated  contract  violently,  draw  in  their  tentacles  or  branchiae, 
and  die  in  a  state  of  contraction  that  renders  the  preserved 
object  a  mere  caricature  of  the  living  animal.  In  these 
cases  special  methods  of  killing  must  be  resorted  to. 

Sudden  Killing. 

11.  Heat. — Speaking   generally,    there    are    two    ways   of 
dealing  with  these  difficult  cases.      You  may  kill  the  animal 
so  suddenly  that  it  has  not  time   to  contract  ;  or  you  may 
paralyse  it  by  narcotics  before  killing  it. 

The  application  of  Heat  is  a  good  means  of  killing 
suddenly.  It  has  the  great  advantage  of  allowing  of  good 
staining  subsequently,  and  of  hindering  less  than  any  other 
method  the  application  of  chemical  tests  to  the  tissues.  By 
it  the  tissues  are  fixed  at  the  same  time  that  somatic  death 
is  brought  about. 

The  difficulty  consists  in  hitting  off  the  right  temperature, 
which  is  of  course  different  for  different  objects.  I  think 


12  CHAPTER   II. 

that  a  temperature  of  80°  to  90°  C.  will  generally  be  amply 
sufficient^  and  that  very  frequently  it  will  not  be  necessary  to 
go  beyond  60  C.  An  exposure  to  heat  for  a  few  seconds  will 
generally  suffice. 

Small  objects  (Protozoa,  Hydroids,  Bryozoa)  may  be  brought  into  a  drop 
of  water  in  a  watch-glass  or  cm  a  slide,  and  heated  over  the  flame  of  a  spirit 
lamp.  For  large  objects,  the  water  or  other  liquid  employed  as  the  vehicle 
of  the  heat  may  be  heated  beforehand  and  the  animals  thrown  into  it. 

As  soon  as  it  is  supposed  that  the  protoplasm  of  the  tissues  is  coagulated 
throughout,  the  animals  should  be  brought  into  alcohol  (30  to  70  per  cent, 
alcohol)  (if  water  be  employed  as  the  heating  agent). 

An  excellent  plan  for  preparing  many  marine  animals  is  to  kill  them  in 
hot  fresh  water.  Some  of  the  larger  Nemertians  are  better  preserved  by 
this  method  than  by  any  other  with  which  I  am  acquainted. 

12.  Slowly  Contracting  Animals. — Animals     that     contract 
but    slowly,   such   as   Alcyonium  and    Veretillum,  and    some 
Tunicates,    such    as    Pyrosoma,    are     very    well    killed    by 
throwing  them  into  some  very  quickly  acting  fixing  liquid, 
either  used  hot  or  cold.      Glacial  or  very  strong  acetic  acid 
(VAN    BEN  EDEN'S   method)    is   an  excellent   reagent   for  this 
purpose  ;  it  may  be  used,  for  example,  with   some  Medusae. 
After  an  immersion  of   a   few    seconds    or   a   few   minutes, 
according  to  the  size  of  the  animals,  they  should  be  brought 
into  alcohol  of  at  least  50  per  cent,  strength.      See  lc  Acetic 
acid  "  and  ' *  Tunicata."      Lemon  juice  employed  in  this  way 
has    given   me  very   good   results  with  small  Annelids   and 
Hirudinea.      Corrosive  sublimate  is  another  excellent  reagent 
for  this  purpose. 

Narcotisation. 

13.  The   secret  of    narcotisation   consists   in   adding   some 
anaesthetic  substance  very  gradually,  in  very  small  doses,  to 
the  water  containing  the  animals,  and  waiting  patiently  for 
it  to  take  effect  slowly. 

The  Tobacco-smoke  Method  for  Actiniae,  due  to  Lo  BIANCO  (Jena 
Zeit.  Naturw.,  Bd.  xiii,  1879,  p.  467;  Mitth.  Zool  Stat.  Neapel,  Bd.  ix, 
1890,  p.  499),  used  to  be  practised  as  follows: — A  dish  containing  the 
animals  in  water  is  covered  with  a  bell-glass,  under  which  passes  a  curved 
glass  or  rubber  tube,  which  dips  into  the  water.  Tobacco  smoke  is  blown 
into  the  water  for  some  time  through  the  tube,  and  the  animals  are  then  left 
for  some  hours.  More  smoke  is  then  blown  in,  and  the  animals  are  left  over- 


KILLING.  13 

night.  Next  morning  they  should  be  irritated  from  time  to  time  by  touch- 
ing  a  tentacle  with  a  needle.  As  soon  as  it  is  observed  that  an  animal 
begins  to  react  slowly — that  is  to  say,  as  soon  as  it  is  found  that  the  contrac- 
tion of  the  tentacle  does  not  begin  until  a  considerable  time  after  it  has 
been  irritated  by  the  needle — the  narcotisation  may  be  considered  sufficient. 
It  may  be  further  completed,  if  desired,  by  the  addition  of  a  little  chloro- 
form. A  quantity  of  some  fixing  liquid  sufficient  to  kill  the  animals  before 
they  have  time  to  contract  is  then  added  to  the  water. 

14.  Nicotin  in  solution  may  be  used  instead  of  tobacco  smoke 
(ANDRKS,  Atti  R.  Accad.  dei  Lincei,  v,  1880,  p.  9  ;  see  Journ. 
Roy.  Mic.  Sue.,  N.  S.,  ii,  1882,  p.  881).      Andres  employs  a 
solution  of  1  gramme  of  nicotin  in  a  litre  of  sea  water.     The 
animal  to  be  anaesthetised  is  placed  in  a  jar  containing  half 
a  litre  of  sea  water,  and  the  solution  of  nicotin  is  gradually 
conducted  into  the    jar  by  means  of    a   thread   acting  as  a 
siphon.      The   thread  ought  to  be  of  such  a  thickness  as  to 
be  capable  of  carrying  over   the  whole  of   the    solution   of 
nicotin  in   twenty-four  hours.      See   also  Mitth.  Zool.  Stat. 
Neapel,  Bd.  ii,  1880,  p.  123. 

15.  Chloroform  may  be  employed  either  in  the  liquid  state 
or  in  the   state   of   vapour.      KOROTNEFP  (Mitth.  Zool.  Stat. 
Neapel,  v,  Hft.  2,  1884,  p.  233)  operates   in  the  following 
manner  with   Siphonophora.      The  animals  being  extended, 
a  watch-glass  containing  chloroform  is  floated  on  the  surface 
of  the  water  in  which  they  are  contained,  and  the  whole  is 
covered  with    a   bell-glass.       As   soon  as  the  animals  have 
become  insensible  they  are  killed  by  means  of  hot  sublimate 
or  chromic  acid  solution  plentifully  poured  on  to  them. 

Liquid  chloroform  is  employed  by  squirting  it  in  small 
quantities  on  to  the  surface  of  the  water  containing  the 
animals.  A  syringe  or  pipette  having  a  very  small  orifice,  so 
as  to  thoroughly  pulverise  the  chloroform,  should  be  employed. 
Small  quantities  only  should  be  projected  at  a  time,  and  the 
dose  should  be  repeated  every  five  minutes  until  the  animals 
are  anaesthetised. 

I  have  seen  large  Medusae  very  completely  anaesthetised  in 
the  state  of  extension  in  an  hour  or  two  by  this  method. 
ANDRES  finds  that  this  plan  does  not  succeed  with  Actiniae, 
a-  \vith  them  maceration  of  the  tissues  supervenes  before 
anaesthesia  is  established. 


14  CHAPTER    II. 

PREYH;R  (Mitth.  Zool.  Stat.  Neapel,  Bd.  vii,  1886,  p.  27)  re- 
commends chloroform  water  for  anaesthetising  star-fishes. 

16.  Ether  and  Alcohol  may  be  administered  in  the  same 
way.    ANDRES  has  obtained  good  results  with  Actiniae  by  the 
use  of  a  mixture  (invented  by  SALVATOEE  LO  BIANCO)  contain- 
ing 20  parts  of   glycerin,  40  parts  of  70  per  cent,  alcohol, 
and  40  parts  of  sea  water.     This  mixture  should  be  carefully 
poured  on  to  the  surface  of  the  water  containing  the  animals, 
.and  allowed  to  diffuse  quietly  through  it.     Several  hours  are 
sometimes  necessary  for  this. 

EISIG  (Fauna  u.  Flora  Golf.  Neapel,  16,  1887,  p.  239) 
benumbs  Capitellidae  by  putting  them  into  a  mixture  of  one 
part  of  70  per  cent,  alcohol  with  9  parts  of  sea  water,  a 
method  which  he  recommends  greatly  for  the  study  of  the 
living  animals. 

17.  Methyl-alcohol.— CORI  (Zeit.  f.  wiss.  Mik.,  vi,  4,  1890, 
p.  438)  prefers  methyl-alcohol  to  all  other  reagents.      It  has 
the  advantage  of    having  but  a  slight  action   on  albumins. 
CORI   recommends   a  mixture   composed    of    10  c.c.  methyl- 
alcohol  (of    96   per    cent,    strength),  90  c.c.  water  (fresh  or 
sea  water),  and  0*6  grm.  of  sodium  chloride  (to  be  added  only 
when  fresh  water  is  taken,  the  addition  of  the  salt  having 
for  its  object  to  prevent   maceration).      It  may  be   well  to 
add  to  this   mixture    a  very    few    drops    of  chloroform    (for 
Cristatella;  Zeit.f.  wiss  Zool.,  Iv,  1893,  p.  626;  Zeit.  f.  wiss. 
Mik.,  x,  4,  1893,  p.  475). 

18,  Hydrate  of  Chloral,  which  WMS  first  recommended,  I 
believe,  by  Foettinger  (Arch,  de  BioL,  vi,  1885,  p.  115),  gives 
very  good  results  with  some  subjects.  Foettinger  operates 
by  dropping  crystals  of  chloral  into  the  water  containing 
the  animals.  For  Alcyonella  he  takes  25  to  80  centigrammes 
of  chloral  for  each  hundred  grammes  of  water.  It  takes 
about  three  quarters  of  an  hour  to  render  a  colony  sufficiently 
insensible  to  allow  of  fixing.  Foettinger  has  obtained  satis- 
factory results  with  marine  and  fresh-water  Bryozoa,  with 
Annelida,  Mollusca,  Nemertians,  Actiniae,  and  with  Asteracan- 
thion.  He  did  not  succeed  with  Hydroids. 


KILLING.  15 

Lo  BIANCO  (Mltth.  Zool.  Stat.  Neapel,  Bd.  ix,  1890,  p.  442) 
employs  for  various  marine  animals  freshly  prepared  solu- 
tions of  chloral  in  sea  water,  of  from  one  tenth  to  one  fifth 
per  cent,  strength. 

I  am  bound  to  state  that  I  have  never  had  the  slightest  success  with  Ne- 
mertians. 

VEEWOEN  (Zeit.  f.  wiss.  Zool.,  xlvi,  1887,  p.  99;  see  also  Journ.  Roy. 
Mic.  Soc.,  1888,  p.  148)  puts  Cristatella  for  a  few  minutes  into  10  per  cent, 
solution  of  chloral,  in  which  the  animals  sooner  or  later  become  extended. 

KUKENTHAL  (Jena  Zeit.  Naturw.,  Bd.  xx,  1887,  p.  511 ;  Journ.  Roy. 
Mic.  Soc.,  1888,  p.  509)  has  obtained  good  results  with  some  Annelids  by 
means  of  a  solution  of  one  part  of  chloral  in  1000  parts  of  sea  water. 

The  chloral  method  gives  rise  to  maceration  with  some 
subjects,  as  I  can  testify,  and  has  been  said  to  distort  nuclear 
figures. 

19.  Cocaine  (RICHARDS,  Zool.  Anz.,   196,  1885,  p.  332)  has 
been  found  to  give  good  results.      Richards  puts  a  colony  of 
Bryozoa  into  a  watch-glass   with  5  c.c.  of  water,  and  adds 
gradually  I  per  cent,  solution  of  hydrochlorate  of  cocaine  in 
water.     After  five  minutes  the  animals  are  somewhat  numbed, 
and  half  a  cubic  centimetre  of   the   solution    is  added  ;  and 
ten  minutes  later  the  animals  should  be  found  to  be  dead  in 
a  state  of  extension. 

This  method  is  stated  to  succeed  with  Bryozoa,  Hydra, 
and  certain  worms.  It  is  the  best  method  for  Rotifers 
(ROUSSKLET).  It  has  also  been  recommended  for  Aplysia. 

It  has  been  pointed  out  (by  GOBI,  in  the  paper  quoted  above)  that  unfor- 
tunately when  fixing  agents,  such  as  sublimate  solution,  are  added  to  the 
animals,  the  cocaine  is  thyown  down  on  them  as  a  white  precipitate.  This 
precipitate,  however,  may  be  redissolved  afterwards  in  alcohol  (EisiG). 

19a.  Menthol  (SoRBY,  Flcreanus,  Sheffield,  1898,  No.  4,  p.  68  ;  Journ 
Roy.  Mic.  Soc.,  1899,  i,p.  103). — Sorby  finds  that  by  adding  a  small  quan- 
tity of  menthol  to  the  sea  water  in  which  "  marine  animals  "  (which  ?)  are 
kept,  they  fully  expand  themselves  and  die  in  a  "  distended  "  condition. 
He  has  thus  succeeded  with  Synapta  and  several  species  of  sea-anemones. 

20.  Hydroxylamin. — HOFEU  (Zeit.  f.  wis*.  Mik.,  vii,  3,  1890, 
p.    318)   has  employed  hydroxylamin  as  a  paralysing  agent 
with  success  with  the  most  varied  animal  forms.      Either  the 
sulphate  or  the  hydrochlorate  of  the  base  may  be  used.      He 
recommends  that  the  hydrochlorate  be  taken.      This,  as  found 


16  CHAPTER    II. 

in  commerce,  is  usually  contaminated  with  HC1.  It  should 
be  dissolved  in  water  (spring  or  sea  water,  according  to  the 
habitat  of  the  organisms — in  no  case  distilled  water),  and  the 
solution  exactly  neutralised  by  addition  of  carbonate  of  soda. 
A  1  per  cent,  solution  should  be  made  up,  and  further  diluted 
for  use.  The  organisms  are  placed  in  the  diluted  solution, 
which  may  be  taken  of  a  strength  varying  from  0*1  percent., 
used  for  thirty  minutes  or  less  (as  for  Infusoria),  to  0*25  per 
cent.,  used  for  from  fifteen  minutes  to  one  hour  (Hydra),  1 
per  cent.,  one  half  to  two  hours  (Hirudo),  or  as  much  as  ten 
to  twenty  hours  (Helix  and  Anodouta). 

It  should  be  remembered  that  hydroxylamin  is  an  extremely 
powerful  reducing  agent.  Care  must  therefore  be  taken  not 
to  treat  the  paralysed  animals  with  easily  reducible  fixing 
agents,  such  as  osmic  acid,  chromic  acid,  sublimate,  chlorides 
of  gold  or  platinum,  etc.,  unless  it  have  been  possible  first  to 
sufficiently  wash  out  the  hydroxylamin  with  water. 

21.  Chloride  of  Magnesium. — TULLBERG  (Arch.  ZooL  Exper.  et  Gen., 
x,  1892,  p.  11 ;  Journ.  Roy.  Hie.  Soc.,  1892,  p.  435)  has  obtained  some 
results  with  this  salt.     For  Actiniae,  a  33  per  cent,  solution  of  the  salt  is  to 
be  very  slowly  added  to  the  water  containing  the  expanded  animal,  until  the 
vessel  contains  1  per  cent,  of  the  salt  (thus  for  one  litre  of  sea  water  33  c.c.  of 
the  solution  must  be  added).     The  addition  must  be  made  gradually,  but  it 
must  be  effected  within  half  an  hour.     Thirty  minutes  later  the  animal  will 
be  found  to  be  anaesthetised,  and  may  be  fixed. 

For  terrestrial    and  fresh-water  Invertebrates  rather  stronger  solutions 
should  be  used. 

EEDKNBAUGH  (Amer.  Natural.,  xxix,  1895,  p.  399  ;  Journ. 
Roy.  Mic.  Soc.,  1895,  p.  385)  has  obtained  good  results  by 
means  of  Sulphate  of  Magnesia,  either  added  in  crystals  to 
the  sea  water  containing  the  animals  until  a  saturated  solu- 
tion is  obtained,  or  in  the  shape  of  a  saturated  solution  into 
which  they  are  thrown  (Annelids). 

GEKAULD  (Bull.  Mus.  Comp.  ZooL,  Harvard,  xxix,  1896, 
p.  123)  has  had  good  results  with  Caudina  arenata  by  means 
of  the  crystals  added  in  teaspoonfuls. 

22.  Poisoning  by  small  doses  of  some  fixing  agent  is  sometimes  a  good 
method.     SALVATOEE  LO  BIANCO  employs  the  following  method  for  pre- 
serving Ascidia  and  Rhopalsea  in  an  extended  state  (Mitth.  Zool.  Stat. 
Neapel,  ix,  1890,  p.  471).     A  little  1  per  cent,  chromic  acid  is  poured  on  to 
the  surface  of  the  water  containing  the  animals,  and  allowed  to  slowly 


KILLING.  17 

diffuse  into  it.  About  twelve  to  twenty-four  hours  is  necessary.  He  kills 
Ciona  in  a  similar  way  with  a  mixture  of  one  part  of  1  per  cent,  chromic 
acid  and  nine  parts  of  49  per  cent,  acetic  acid. 

Osmic  acid,  or  Kleinenberg's  solution,  is  sometimes  employed  in  the 
same  way. 

I  have  seen  Medusae  killed  in  a  satisfactory  manner  by  means  of  crystals 
of  corrosive  sublimate  added  to  the  water  containing  them. 

Morphia,  Curare,  Strychnin,  Prussic  Acid,  and  other  paralysing 
drugs  have  also  been  employed. 


23.  Asphyxiation  may  be  sometimes  successfully  practised. 
Terrestrial  Gastropods  may  be  killed  for  dissection  by  putting 
them  into  a  jar  quite  full  of  water  that  has  been  deprived  of 
its  air  by  boiling,  and  hermetically  closed.  After  from 
twelve  to  twenty-four  hours  the  animals  are  generally  found 
dead  and  extended.  The  effect  is  obtained  somewhat  quicker 
if  a  little  tobacco  be  added  to  the  water. 

Good  results  are  sometimes  obtained  with  aquatic  animals 
by  simply  leaving  them  to  exhaust  the  oxygen  of  the  water  in 
which  they  are  contained.  I  have  sometimes  succeeded  with 
Holothuriaa  and  other  Echinoderms  in  this  way.  WARD  (see 
Amer.  Nat.,  xxv,  1891,  p.  398)  has  succeeded  with  Hydroids, 
Actiniae,,  and  similar  forms,  and  UEXKULL  (Mitth.  Zool.  Stat. 
Neupel,  xii,  1896,  p.  468)  with  Echinids.  If  the  animals  be 
found  to  be  imperfectly  expanded  when  narcosis  has  set  in, 
they  may  be  got  to  expand  by  putting  them  back  for  a  short 
time  into  pure  sea  water ;  and  as  soon  as  they  are  expanded 
should  be  quickly  thrown  into  some  rapidly  killing  reagent. 

Marine  Animals  are  sometimes  successfully  killed  by  simply 
putting  them  into  fresh  water. 

Warm  Water  will  sometimes  serve  to  immobilise  and  even 
kill  both  marine  and  fresh-water  organisms. 

24.  Carbonic  Acid  Gas  has  been  recommended  (by  FOL,  Zool. 
Anz.,,  128,  1885,  p.  698).  The  water  containing  the  animals 
should  be  saturated  with  the  gas.  The  method  is  stated  to 
succeed  with  most  Coelenterata  and  Echinodermata,  but  not 
with  Molluscs  or  Fishes.  I  have  had  most  excellent  results 
with  small  Annelids  and  Hirudinea.  It  is  not  necessary  to 
employ  a  generator  for  obtaining  the  gas.  It  suffices  to  take 
an  ordinary  "  soda-water "  siphon,  and  squirt  its  contents 
into  the  water  containing  the  animals. 

2 


18  CHAPTER    II. 

Narcotisation  is  very  rapidly  obtained  with  very  small 
animals,  but  much  more  slowly  with  larger  ones.  For 
instance,  Stylaria  proboscidea,  I  find,  is  paralysed  in  a  few 
seconds  •  a  small  Nephelis,  of  15  or  20  millimetres  in  length, 
will  require  about  five  minutes;  and  a  large  Nephelis,  of 
from  10  to  15  centimetres,  will  require  as  many  hours.  Nar- 
cotised animals  recover  very  quickly  on  being  put  back  into 
pure  water. 

UEXKULL  (Mitth.  Zool.  Stat.  Neapel,  xii,  1896,  p.  463)  has 
paralysed  Echinids  very  rapidly  with  carbonic  acid,  likewise 
a  small  Teleostean  fish;  whilst  Scyllium  and  Crustaceans 
were  affected  much  more  slowly,  and  mussels  not  at  all. 

25.  Peroxide  of  Hydrogen. — VOLK  (Zool.  Anz.,  Bd.  xix,  1896, 
p.  294)  kills  Eotatoria  by  means  of  one  or  two  drops  of  a 
3  per  cent,  solution  added  to  1  c.c.  of  the  water  containing 
them.  If  the  right  dose  has  been  hit  off  they  die  extended, 
and  are  then  brought  first  into  pure  water  and  then  into 
some  fixing  liquid. 


CHAPTER   III. 

FIXING  AND   HARDENING. 

26.  The  Functions  of  Fixing  Agents. — The  meaning  of  the 
term  "  fixing  "  has  been  explained  above  (§  2).  It  remains 
here  to  insist  on  the  absolute  necessity  of  the  employment 
of  fixing  agents,  and  to  briefly  illustrate  this  necessity.  If  a 
portion  of  living  retina  be  placed  in  aqueous  humour,  serum, 
or  other  so-called  "  indifferent "  medium,  or  in  any  of  the 
media  used  for  permanent  preservation,  it  will  be  found  that 
the  rods  and  cones  will  not  preserve  the  appearance  they 
have  during  life  for  more  than  a  very  short  time  ;  after  a  few 
minutes  a  series  of  changes  begins  to  take  place,  by  which 
the  outer  segments  of  both  rods  and  cones  become  split  into 
discs,  and  finally  disintegrate  so  as  to  be  altogether  unrecog- 
nisable, even  if  not  totally  destroyed.  Further,  in  an 
equally  short  time  the  nerve-fibres  become  varicose,  and 
appear  to  be  thickly  studded  with  spindle-shaped  knots  ;  and 
other  post-mortem  changes  rapidly  occur.  If,  however,  a 
fresh  piece  of  retina  be  treated  with  a  strong  solution  of 
osmic  acid,  the  whole  of  the  rods  and  cones  will  be  found 
perfectly  preserved  after  twenty-four  hours'  time,  and  the 
nerve- fibres  will  be  found  not  to  be  varicose.  After  this 
preliminary  hardening,  portions  of  the  retina  may  be  treated 
with  water  (which  would  be  ruinous  to  the  structures  of  a 
fresh  retina) ,  they  may  even  remain  in  water  for  days  without 
harm  ;  they  may  be  stained,  acidified,  hardened,  imbedded, 
cut  into  sections,  and  mounted  in  either  aqueous  or  resinous 
media  without  suffering. 

This  example  shows  that  one  of  the  objects  aimed  at  in 
fixing  is  to  impart  to  tissues  the  degree  of  hardening  neces- 
-;n-\  to  enable  them  to  offer  such  mechanical  resistance  to 
post-mortem  change  and  to  the  processes  of  after-treatment 
as  not  to  suffer  change  of  form.  Another  important  func- 


20  CHAPTEli    II  r. 

tion  of  fixing  is  to  render  insoluble  elements  of  cells  and  tissues 
that  would  otherwise  be  more  or  less  dissolved  out  by  the 
liquids  employed  during  the  after-treatment.  Compare  in 
this  respect  the  aspect  of  sections  of  a  piece  of  testis  that 
has  been  well  fixed  in  liquid  of  Flemming  and  cut  in  paraffin, 
with  the  aspect  of  paraffin  sections  of  a  piece  of  the  same 
testis  that  has  not  been  fixed,  or  that  has  only  been  fixed  by 
some  reagent  inadequate  for  the  purpose,  such  as  alcohol  or 
picric  acid.  Jn  the  one  case,  plump,  full,  unshrunken  cells, 
free  from  vacuoles,  fall  of  structure ;  in  the  other,  lean, 
empty,  shrunken  cells,  with  foamy  and  vacuolated  proto- 
plasm, half  their  original  structure  lost,  and  that  which 
remains  distorted  !  Their  appearance,  compared  with  that 
of  living  or  well-preserved  cells,  suggests  at  once  that  much 
must  have  been  dissolved  out  of  them. 

A  third  and  highly  important  function  of  fixing  agents 
consists  in  producing  optical  differentiation  in  structures. 
By  coagulating  the  elements  of  tissues  and  cells,  fixing  agents 
alter  their  indices  of  refraction,  raising  them  in  varying 
degrees.  They  do  not  act  in  an  equal  degree  on  all  the  con- 
stituent elements  of  cells  and  tissues,  but  raise  the  index  of 
some  more  than  that  of  others,  thus  producing  optical 
differentiation  where  there  was  little  or  none  before.  Com- 
pare the  aspect  of  the  epithelium  of  the  tail  of  a  living 
tadpole,  observed  in  water,  with  its  aspect  after  the  action 
of  a  little  diluted  solution  of  Flemming.  In  the  living 
state  the  protoplasm  of  its  cells  has  a  refractive  index  little 
superior  to  that  of  water,  and  consequently  so  low  an  index 
of  visibility  that  hardly  any  structure  can  be  made  out  in  the 
object.  But  as  soon  as  the  protoplasm  has  been  sufficiently 
coagulated  by  the  reagent  the  refractive  indices  of  some  of 
its  elements  will  have  been  raised  to  above  that  of  balsam, 
the  chromatin  of  the  nuclei  will  be  brought  out,  and  other 
structure  be  revealed  where  none  was  visible  before. 

The  notion  of  fixing  is  distinct  from  that  of  hardening. 
All  fixing  presupposes  a  certain  degree  of  hardening,  as 
explained  above.  But  it  does  not  include  the  degree  of 
hardening  necessary  to  give  to  soft  tissues  a  consistency 
which  will  allow  them  to  be  cut  into  thin  sections  without 
imbedding.  This  is  hardening  proper  (see  §  31).  Of  course, 
if  the  stage  of  fixing  be  prolonged,  with  a  view  to  procuring 


FIXING    AND    HARDENING.  21 

enhanced    optical   differentiation,    hardening  will   be   super- 
induced, and  the  one  stage  will  run  into  the  other. 

27.  The  Action  of  Fixing  Agents  consists  in  coagulating 
and  rendering  insoluble  certain  of  the  constituents  of  tissues. 
This  is  effected  sometimes  without  any  chemical  action  being 
involved,  as  when  alcohol  is  employed,  which  acts  by  simple 
withdrawal  of  the  water  of  the  tissues.  But  in  the  majority 
of  cases  the  fixing  agents  enter  into  chemical  combination 
with  certain  of  the  elements  of  the  tissues.  The  compounds 
thus  formed  are  sometimes  unstable  and  soluble,  so  that  they 
are  removable  by  washing,  as  is  the  case  with  several  of 
those  formed  by  picric  acid.  It  is  found  in  practice,  how- 
ever, that  those  formed  by  chromic  acid  and  its  salts,  and 
the  salts  of  the  heavy  metals,  as  mercury,  iron,  platinum, 
gold,  and  silver,  are  mostly  insoluble  ;  but  unfortunately  we 
have  no  exact  chemical  knowledge  of  them. 

The  insolubility  of  these  bodies  is  an  advantage  from  the 
point  of  view  explained  in  the  last  section,  in  that  it  ensures 
that  the  tissues  shall  not  be  robbed  of  their  essential  con- 
stituents, nor  deprived  of  their  desired  consistency  and 
optical  differentiation,  by  the  reagents  subsequently  em- 
ployed. It  is  also  sometimes  an  advantage  in  that  certain 
of  the  compounds  in  question  have  the  property  of  combining 
with  certain  colouring  matters,  and  thus  affording  important 
stains  which  could  not  otherwise  be  obtained  ;  or  in  other 
words,  of  acting  as  mordants. 

But  it  is  sometimes  a  disadvantage,  inasmuch  as  these 
same  compounds  which  render  possible  the  production  of 
some  stains  are  hindrances  to  the  production  of  others. 
Tissues  that  have  been  fixed  with  osmic  or  chromic  acid 
or  its  salts  are  in  general  not  easily  to  be  stained  with 
carmine  or  similar  colouring  matters,  unless  the  metals  have 
been  previously  removed  by  special  chemical  treatment  (see 
§  40,  and  BLEACHING)  ;  though  they  may  generally  be  stained 
with  haemalum,  or,  after  sectioning,  with  iron  hasmatoxylin 
or  tar  colours. 

27a.  Fixation  Images  and  Precipitates.  —  According  to 
FISCHER  (Fixirung,  Farbuny,  und  Ban  des  Protoplasmas ,  Jena, 
G.  Fischer,  1899,  pp.  x,  362)  the  coagulation  which  con- 
stitutes fixation  is,  in  the  case  of  the  liquid  and  semi-liquid 


22  CHAPTER    III. 

constituents  of  tissues,  always  a  phenomenon  of  precipitation. 
The  more  solid  constituents  (such  as  fibrils  that  are  visible 
during  life,  nucleoli,  and  the  like)  he  admits  may  be  acted  on 
by  fixing  reagents  without  the  formation  of  any  visible  pre- 
cipitates. But  all  the  liquid  ones,  in  so  far  as  they  are 
fixed  at  all,  are  visibly  precipitated  in  special  precipitation 
forms,  which  vary  according  to  the  precipitant.  Each  fixing 
agent  gives  its  own  characteristic  fixation  image,  which  may 
be  more  or  less  lifelike,  but  can  never  be  absolutely  so. 
Fischer  gives  copious  descriptions  of  the  precipitation 
forms  of  the  chief  organic  compounds  found  in  tissues,  and 
of  the  precipitation  powers  of  the  chief  fixing  agents,  which 
the  reader  will  do  well  to  study. 

It  seems  to  be  a  consequence  of  Fischer's  theory  of  fixa- 
tion by  precipitation  that  the  most  energetic  fixing  agents 
should  always  be  found  amongst  the  most  energetic  precipi- 
tants.  But  on  the  showing  of  his  experiments  this  is  not  so. 
For  instance,  it  is  allowed  on  all  hands  that  osmic  acid  is  a 
most  energetic  fixative.  But  Fischer  finds  (op.  cit.,  pp.  12 
—14,  27)  that  it  is  a  very  incomplete  and  weak  precipitant. 
Or,  to  take  a  contrary  instance,  he  finds  that  picric  acid  is 
an  energetic  precipitant  of  the  majority  of  cell  constituents ; 
but  surely  every  cytologist  must  admit  that  it  is  a  very 
incomplete  fixative. 

It  would  seem  to  follow,  from  these  instances  and  from 
other  similar  ones,  that  Fischer 's  tables  of  precipitating  power 
cannot  be  taken  as  a  measure  of  the  fixing  power  of  the 
reagents.  And  further,  the  study  of  the  fixation  images  of 
tissues  afforded  by  osmic  acid,  formaldehyde,  and  other 
reagents,  seems  to  show  that  the  coagulation  brought  about 
by  them  is  in  part  accompanied  by  the  formation  of  pre- 
cipitates, but  in  part  not  so,  and  that  they  may  do  their 
work  to  a  larger  extent  than  he  seems  to  admit  through  a 
homogeneous  coagulation.  But  from  his  very  suggestive 
observations  and  reasonings  it  certainly  appears  that  the 
formation  of  visible  precipitates  is  a  very  wide-spread,  if  not 
universal  concomitant  of  fixation  ;  and  that  the  wider  the 
precipitating  power  of  a  fixative  (i.  e.  the  greater  the  number 
of  organic  liquids  that  it  can  precipitate),  the  greater  will  be 
the  number  of  artefacts  to  which  it  can  give  rise.  His  work 
is  deserving  of  most  careful  study. 


FIXING   AND    HARDENING.  23 

28.  The  Characters  of  the  Usual  Fixing  Agents. — No  single 
substance  or  chemical  compound  fulfils  all  that  is  required 
of  a  good  fixing  agent ;  hence  it  is  that  without  exception 
all  the  best  fixing  agents  are  mixtures.  A  good  fixing  agent 
should  first  of  all  preserve  all  the  elements  it  is  desired  to 
fix.  But  that  is  not  enough ;  it  should  also  give  good 
optical  differentiation,  and  should  have  sufficient  power  of. 
penetration  to  ensure  that  small  pieces  of  tissue  be  equally 
fixed  by  it  throughout.  Osmic  acid,  which  is  one  of  the 
finest  fixatives  known,  fulfils  some  of  these  conditions,  but 
not  all  of  them.  It  kills  rapidly,  and  preserves  admirably 
the  elements  of  cytoplasm,  but  nuclei  not  so  well.  But  the 
optical  differentiation  that  it  gives,  though  sometimes  good, 
is  often  very  inferior.  For  osmic  acid,  by  coagulating  in 
nearly  equal  degrees  alike  spongioplasm  (the  plastin  reticu- 
lum)  and  hyaloplasm  (the  enchylema)  and  the  chromatin  of 
nuclei,  raises  alike  the  refractive  indices  of  all  of  them ;  so 
that  if  the  fixing  action  have  been  in  the  least  degree  over- 
done, the  cells  acquire  a  homogeneous  aspect  in  which  the 
finer  details  are  obscured  by  the  general  refractivity  of  the 
whole.  If  now,  instead  of  using  it  pure,  it  be  used  in  com- 
bination with  acetic  acid,  a  better  differentiation  is  obtained ; 
Tor  acetic  acid  is  properly  a  fixative  only  for  a  limited  time, 
whilst,  if  its  operation  be  prolonged,  it  exercises  a  swelling 
and  solvent  action  on  the  elements  of  protoplasm.  It  there- 
fore, whilst  enhancing,  or  at  all  events  not  interfering  with 
the  fixation  of  the  chromatin,  serves  to  facilitate  penetration 
and  to  counteract  the  excessive  action  of  the  osmic  acid  on 
the  protoplasm,  so  that  the  cells  come  out  less  homogeneous 
and  with  more  detail  observable  in  them.  A  still  better 
effect  is  obtained  if  to  the  osmic  acid  there  be  added  not  only 
acetic  acid,  but  also  chromic  acid.  For  osmic  acid  has  the 
property  of  blackening  tissues,  thus  rendering  them  opaque. 
Chromic  acid  counteracts  in  a  considerable  degree  this 
blackening  action.  It  also  helps,  probably,  to  bring  out  the 
chromatin  of  nuclei,  which  is  insufficiently  fixed  by  the  other 
two  ingredients,  and  perhaps  also  to  counteract  the  exces- 
sive coagulation  of  hyaloplasm  by  the  osmic  acid  ;  so  that  in 
the  result  a  much  clearer  picture  is  obtained. 

Such  a  mixture  gives  admirable  results  so  far  as  preser- 
vation and  differentiation  are    concerned.      But    as   regards 


24  CHAPTER    III. 

penetration  it  gives  only  very  bad  results.  Osmic  acid  is 
hopelessly  deficient  in  power  of  penetration,  and  no  admixture 
with  other  substances  has  been  successful  in  curing  this 
defect.  So  that  whenever  a  fair  degree  of  penetration  is 
required,  some  other  reagent  must  be  resorted  to.  Picric 
acid  is  one  of  the  most  penetrating  fixatives  knowu,  but  its 
hardening  power  is  very  slight,  so  that  in  order  to  produce 
the  best  results  it  ought  to  be  combined  with  some  more 
energetically  hardening  reagent.  Corrosive  sublimate  is 
very  penetrating  and  hardens  very  energetically,  but  in  no 
form  in  which  I  have  tried  it  does  it  give  the  fine  optical 
differentiations  that  are  obtained  by  means  of  the  osmic 
mixtures.  Bichromate  of  potash  is  an  admirable  preserva- 
tive of  protoplasm,  but  is  not  very  penetrating,  and  does  not 
properly  preserve  the  chromatin  of  nuclei,  causing  it  to 
swell.  This  defect  may  be  overcome  by  combining  it  with 
sufficient  acetic  acid  ;  but  the  defect  of  want  of  penetration 
will  still  remain. 

I  take  it  that  it  has  been  established  by  experience  that, 
as  a  general  rule,  in  order  to  get  the  best  results,  all  fixatives 
should  have  an  add  reaction.  Consequently,  if  their  chief 
ingredients  have  not  naturally  an  acid  reaction,  they  should 
be  acidified,  e.  g.  osmic  acid  should  be  acidified  with  acetic 
acid  or  the  like.  As  a  matter  of  fact,  it  will  be  found 
that  acetic  acid  is  very  largely  employed  in  mixtures. 
It  is  generally  held  that  it  acts  in  them  as  an  agent 
for  facilitating  penetration  and  producing  differentiation, 
as  explained  above,  and  also  for  ensuring  the  fixation  of 
nuclein  (if  the  other  ingredients  are  not  adequate  thereto, — as, 
for  example,  in  the  case  of  bichromate  of  potash)  ;  but  this 
is  probably  not  all.  FJSCHER  (in  the  work  quoted  §  2 7 a, 
pp.  10,  27,  and  other  places)  holds  that  its  function  in 
these  mixtures  is  chiefly  that  of  a  neutralises'  or  addifier 
(Ansaurer)  for  ensuring  that  the  other  ingredients  shall  have 
an  acid,  or  at  least  a  neutral  medium  to  do  their  work  in. 
For  the  precipitating  power,  that  is  in  his  view  the  fix- 
ing power  of  a  reagent,  varies  according  to  the  reaction, 
acid  or  alkaline,  of  the  things  to  be  fixed ;  and  a  weakly 
acid  reaction  is  the  one  most  favourable  for  ensuring  pre- 
cipitation. Many  things  that  are  quite  unprecipitable  by 
certain  reagents  whilst  in  alkaline  or  neutral  solution  are 


FIXING   AND   HARDENING.  25 

immediately  precipitated  by  them  if  the  solution  is  rendered 
acid.  "  Many  kinds  of  cell  contents/'  he  says  (op.  cit., 
p.  10)  "  indeed  the  majority,  have  an  alkaline  reaction,  and 
are  thereby  quite  inaccessible  to  the  precipitating  action  of 
certain  agents,  such  as  osmic  acid,  or  bichromate  ;  and  the 
action  of  certain  other  fixatives,  such  as  platinum  chloride 
and  chromic  acid,  is  more  or  less  hindered  by  the  presence 
of  free  alkalies.  For  neither  the  chromic  acid  (of  solution  of 
F lemming)  nor  the  platinum  chloride  (of  solution  of  Hermann) 
would  be  adequate  to  act  as  acidifiers  to  the  osmic  acid  of 
the  mixtures.  They  cannot  do  so,  firstly,  because  they  them- 
selves become  combined  with  the  tissues  much  more  quickly 
than  the  slowly  working  osmic  acid,  and  secondly,  because 
they  themselves  have  only  an  extremely  weak  acid  reaction/7 
Hence  the  function  of  the  organic  acid  is  to  bring  into  play 
the  precipitating  power  of  the  other  ingredients. 

Not  only  is  it  true  that  the  most  complete  fixations  can 
only  be  obtained  by  means  of  mixtures,  but  it  is  also  true 
that  no  one  mixture  can  serve  all  ends.  It  is  probably  mis- 
leading to  recommend  this  or  that  reagent  as  "  the  best  for 
general  purposes,"  or  the  like.  The  following  suggestions, 
however,  may  be  helpful  to  the  beginner. 

In  Part  II  will  be  found  mentioned  the  fixatives  that 
appear  the  most  appropriate  for  different  purposes  (embryo- 
logical,  cytological,  etc.),  and  for  the  different  tissues  and 
organs,  etc. 

Speaking  generally,  I  think  the  beginner  should  avoid 
such  things  as  liquid  of  FLEMMING  and  similar  mixtures.  He 
may  take,  instead,  liquid  of  TELLYESNICZKY.  This  gives  a  fair 
fixation  and  is  easy  to  manage,  but  it  is  wanting  in  pene- 
tration. 

( 'ormsive  sublimate  is  a  good  all-round  fixative,  with 
excellent  penetration,  but  is  not  quite  so  easy  to  manage. 

Picric  acid  gives  a  fair  though  weak  fixation,  with  very 
good  penetration,  is  easy  to  manage,  and  does  not  make 
ti><ues  brittle,  which  sublimate  easily  may  do. 

To  ensure  the  best  results,  all  fixative*  should  be  acid,  for 
the  reasons  explained  above.  They  may  conveniently  be 
made  to  contain  from  one  to  five  per  cent,  of  acetic  acid. 

The  student  may  consult  with  advantage,  besides  the  work 
of  FISCHEK  quoted  §  27 'a,  the  paper  of  TELLYESNICZKY,  Arch. 


26  CHAPTER    III. 

/.  mik.  Anat.,  lii,  2,  1898,  "  Ueber  die  Fixirungs-  (Hart- 
ungs-)  Fliissigkeiten/'  and  that  of  WASIELEWSKI,  Zeit.f.  wiss. 
Mik.}  xvi,  3,  1899,  "  Ueber  Fixirungsfliissigkeiten  in  der 
botanischen  Mikrotechnik."  The  results  of  these  authors  are 
in  the  main  in  agreement  with  my  own,  but  not  entirely. 
The  points  of  difference  are  probably  to  a  great  extent  to  be 
explained  by  the  fact  of  these  observers  having  each  of  them 
confined  his  attention  to  a  single  category  of  cells.  Now  it 
is  by  no  means  true  that  that  which  fixes  one  sort  of  cells 
well  or  ill  will  also  fix  the  majority  of  others  in  a  similar 
way. 

29.  The  Practice  of  Fixation. — Hints  and  Cautions. — See  that 
the  structures  are  perfectly  living  at  the  instant  of  fixation, 
otherwise  you  will  only  fix  pathological  states  or  post-mortem 
states. 

Do  all  you  can  to  facilitate  the  rapid  penetration  of  the 
•fixing  agent.  To  this  end  let  the  structures  be  divided  into 
the  smallest  portions  that  can  conveniently  be  employed, 
and  if  entire  organs  or  organisms  are  to  be  fixed  whole,  let 
openings,  as  large  as  possible,  be  first  made  in  them. 

The  penetration  of  reagents  is  greatly  facilitated  by  heat. 
You  may  warm  the  reagent  and  put  it  with  the  object  to  be 
fixed  in  the  paraffin  stove,  or  you  may  even  employ  a  fixing 
agent  heated  to  boiling-point  (as  boiling  sublimate  solution 
for  certain  corals  and  Hydroids,  or  boiling  absolute  alcohol 
for  certain  Arthropods  with  very  resistent  integuments). 
But  this  should  only  be  done  as  a  last  resource. 

Let  the  quantity  of  fixing  agent  employed  be  at  least  many 
times  the  volume  of  the  objects  to  be  fixed.  If  this  precaution 
be  not  observed  the  composition  of  the  fixing  liquid  may  be 
seriously  altered  by  admixture  of  the  liquids  or  of  the  soluble 
substances  of  the  tissues  thrown  into  it.  For  a  weak  and 
slowly  acting  fixing  agent,  such  as  picric  acid,  the  quantity  of 
liquid  employed  should  be  in  volume  about  one  hundred  times 
that  of  the  object  to  be  fixed.  Reagents  that  act  very  ener- 
getically, such  as  Flemming's  solution,  may  be  employed  in 
smaller  proportions. 

BBATJS  and  DRUENER  (Jena.  Zeit.  Naturw.,  Bd.  xxix,  1895,  p.  435)  fix 
fishes  ~by  injection  through  the  bulbus  aortx.  The  vessels  are  first  washed 
out  with  normal  salt  solution,  the  fixing  liquid  is  then  thrown  in,  then  as 


FIXING   AND    HARDENING.  27 

soon  as  fixation  is  judged  to  be  complete  water  is  injected ;  lastly,  alcohol, 
and  the  fishes  are  thrown  into  alcohol.  Or,  if  chromic  liquids  have  been 
employed,  the  fishes  are  put  direct  into  solution  of  Miiller.  See  also  the 
methods  of  fixation  by  injection  of  GOLGI,  DE  QUERVAIN,  and  MANX,  given 
under  Neurological  Methods. 

As  regards  the  time  during  which  fixing  reagents  should 
be  allowed  to  act,  it  is  well,  as  a  general  rule,  not  to  leave 
specimens  in  them  for  a  longer  time  than  is  sufficient  to 
obtain  the  desired  reaction.  Sublimate,  for  instance,  soon 
makes  tissues  brittle.  But  long  immersion  may  be  necessary 
to  produce  the  desired  optical  differentiation  with  some 
reagents ;  and  I  now  think  that  the  school  of  Flemming,  who 
sometimes  leave  tissues  for  many  weeks  in  solution  of 
Flemming,  may  be  right  in  their  practice,  for  the  special 
objects  that  they  have  in  view. 

Careful  washing  out  (by  which  is  meant  the  removal  from 
the  tissues  of  the  excess  of  uncombined  fixative)  is  necessary 
in  order  to  get  tissues  to  stain  properly.  But  it  is  not  always 
equally  imperative.  Alcohol  and  formaldehyde  do  not  require 
washing  out  before  staining ;  acetic  and  picric  acid  only  for 
some  stains  ;  sublimate  will  allow  of  staining  even  if  not 
washed  out,  but  allows  of  a  sharper  stain  if  well  washed  out : 
all  osmic,  chromic,  and  platinic  liquids  require  very  thorough 
washing  out. 

Be  careful  to  use  the  appropriate  liquid  for  washing  out 
the  fixing  agent  after  fixation.  It  is  frequently  by  no  means 
a  matter  of  indifference  whether  water  or  alcohol  be  employed 
for  washing  out.  Sometimes  water  will  undo  the  whole  work 
of  fixation  (as  with  picric  acid).  Sometimes  alcohol  causes 
precipitates  that  may  ruin  the  preparations.  Instructions  on 
this  head  are  given  where  necessary. 

Use  liberal  quantities  of  liquid  for  washing. 

Change  the  liquid  as  often  as  it  becomes  turbid,  if  that 
should  happen. 

The  process  of  washing  out  is  often  greatly  facilitated  by 
heat.  Picric  acid,  for  instance,  is  nearly  twice  as  soluble  in 
alcohol  warmed  to  40°  C.  as  in  alcohol  at  the  normal  tempe- 
rature (Fol). 

30.  Fixation  of  Marine  Animals. — In  the  case  of  marine 
organisms  it  may  be  stated  as  a  general  rule  that  their  tissues 
are  more  refractory  to  the  action  of  reagents  than  are  the 


28  CHAPTER    III, 

tissues  of  corresponding  fresh-water  or  terrestrial  forms,  and 
fixing  solutions  should  in  consequence  be  stronger  (about  two 
to  three  times  stronger,  according  to  Langerhans). 

Marine  animals  ought  to  be  freed  from  the  sea  water  adhe- 
rent to  their  surface  before  treating  them  either  with  alcohol 
or  any  fixing  reagent  that  precipitates  the  salts  of  sea  water. 
If  this  be  not  done,  the  precipitated  salts  will  form  on  the 
surfaces  of  the  organisms  a  crust  that  prevents  the  penetra- 
tion of  reagents  to  the  interior,  thus  allowing  maceration  to 
be  set  up,  and  hindering  the  penetration  of  staining  fluids. 
Fixing  solutions  for  marine  organisms  should  therefore  be 
such  as  serve  to  keep  in  a  state  of  solution,  and  finally 
remove,  the  salts  in  question.  As  a  general  rule,  they 
should  not  be  made  with  sea  water  as  a  menstruum,  as  some 
workers  have  inconsiderately  proposed.  If,  however,  for 
any  particular  purpose,  it  is  deemed  desirable  so  to  prepare 
them,  care  should  be  taken  to  remove  the  sea  salts  after- 
wards by  appropriate  washing,  or  to  mount  the  objects  in 
glycerin  (MAYEK).  If  alcohol  be  employed,  it  should  be 
acidified  with  hydrochloric  or  some  other  appropriate  acid. 
Picro-nitric  acid  is  a  fixing  reagent  that  fulfils  the  conditions 
here  spoken  of.  (On  this  subject  see  Paul  Mayer,  in  Mitth. 
ZooL  Stut.  Neapel,  ii  (1881),  p.  1,  et  seq.  See  also  the 
abstract  in  Jouni.  Roy.  Hie.  Soc.  (N.  S.),  ii  (1882),  pp.  866— 
881,  and  that  in  AHH.T.  Natural.,  xvi  (1882),  pp.  697—706.) 

'31.  Hardening. — The  process  of  hardening  was  above  (§  26) 
distinguished  from  that  of  fixing  as  being  directed  to  the 
attainment  of  a  degree  of  consistency  sufficient  to  allow  of 
soft  tissues  being  cut  into  sections  without  imbedding.  It 
is  also  distinguished  from  fixing  in  that  it  does  not  include 
the  killing  of  the  elements.  Nerve  tissue,  for  instance,  is 
daily  hardened  after  having  come  into  the  hands  of  the 
anatomist  some  twelve  or  twenty-four  hours  after  the  death 
of  the  subject,  under  which  conditions  there  can  of  course 
be  no  question  of  fixing.  Hardening  is  an  after  process,  and 
only  ranks  as  a  tf^eciiil  method. 

Methods  of  imbedding  have  now  been  brought  to  such  a 
degree  of  perfection  that  the  thorough  hardening  of  soft 
tissues  that  was  formerly  necessary  in  order  to  cut  thin 
sections  from  them  is,  in  the  majority  of  cases,  no  longer 


FIXINO    AND    HARDENING.  29 

necessary ;  by  careful  infiltration  with  paraffin  or  other  good 
infiltration  mass,  most  soft  objects  can  be  satisfactorily  cut 
with  no  greater  an  amount  of  previous  hardening  than  is 
furnished  by  the  usual  passing  of  the  tissues  after  fixing 
through  successive  alcohols  in  order  to  prepare  them  for  the 
paraffin  bath.  But  there  are  some  exceptions.  Such  are, 
for  instance,  the  cases  in  which  it  is  desired  to  cut  very  large 
sections,  such  as  sections  of  the  entire  human  brain.  Such 
an  organ  as  this  cannot  be  duly  infiltrated  with  alcohol  in  a 
few  hours,  and  it  is  doubtful  whether  it  can  be  duly  infiltrated 
with  paraffin  or  any  other  imbedding  mass  in  any  reasonable 
time.  And  certain  organs  that  are  either  extremely  delicate 
or  inaccessible,  such  as  retina  or  cochlea,  will  require  to  be 
specially  hardened  in  order  to  give  the  best  results.  The 
processes  employed  for  hardening  such  specimens  as  these 
will  be  described  when  treating  of  the  organs  in  question. 

The  reagents  employed  for  hardening  are  for  the  most 
part  of  the  same  nature  as  those  employed  for  fixing.  But 
it  does  not  follow  that  all  fixing  agents  can  be  employed  for 
hardening.  Corrosive  sublimate,  for  instance,  would  be 
most  inappropriate  as  a  hardening  agent. 

[The  chapter  on  Hardening  Agents  that  has  appeared  in 
all  former  editions  is  suppressed,  its  contents  having  been 
incorporated  with  the  chapters  on  Fixing  Agents  and  on 
Neurological  Methods.] 

32.  The  Practice  of  Hardening — Hints  and  Cautions. — Employ 
in  general  a  relatively  large  volume  of  hardening  liquid,  and 
change  it  very  frequently.  The  exact  proportions  may  be 
made  out  by  experiment  for  each  reagent  and  each  class  of 
objects.  If  the  volume  of  liquid  be  insufficient  its  composi- 
tion will  soon  become  seriously  altered  by  the  diffusion  into 
it  of  the  soluble  substances  of  the  tissues  ;  and  the  result 
may  be  a  macerating  instead  of  a  hardening  liquid.  Further, 
as  soon  as,  in  consequence  of  this  diffusion,  the  liquid  has 
acquired  a  composition  similar  in  respect  of  the  proportions 
of  colloids  and  crystalloids  contained  in  it  to  that  of  the 
liquids  of  the  tissues,  osmotic  equilibrium  will  become  estab- 
lished, and  diffusion  will  cease  ;  that  is  to  say,  the  harden- 
ing liquid  will  cease  to  penetrate.  This  means,  of  course, 
maceration  of  internal  parts.  On  the  other  hand,  it  appears 


30  CHAPTER    III. 

that  a  certain  slight  proportion  of  colloids  in  the  hardening" 
liquid  is  favourable  to  the  desired  reaction,  as  it  gives  a 
better  consistency  to  the  tissues  by  preventing  them  from 
becoming  brittle.  Hence  the  utility  of  employing  a  certain 
proportion  of  hardening  agent. 

Hardening  had  better  be  done  in  tall  cylindrical  vessels, 
the  objects  being  suspended  by  a  thread,  or  muslin  bag,  or 
otherwise,  at  the  top  of  the  liquid.  This  has  the  advantage 
of  allowing  diffusion  to  take  place  as  freely  as  possible, 
whilst  any  precipitates  that  may  form  fall  harmlessly  to  the 
bottom. 

In  general  begin  hardening  with  a  weak  reaaent,  increasing* 
the  strength  gradually,  as  fast  as  the  tissues  acquire  a  consist- 
ence that  enables  them  to  support  a  more  energetic  action 
of  the  reagent. 

Let  the  objects  be  removed  from  the  hardening  fluid  a 
soon  as  they  have  acquired  the  desired  consistency. 


CHAPTER  IV. 

FIXING    AND   HARDENING  AGENTS MINERAL   ACIDS    AND 

THEIR    SALTS. 

33.  Osmic   Acid. — The  tetroxide  of  osmium   (Os 04)    is  the 
substance   commonly  known    as   osmic  acid,  though    it  does 
not   possess   acid   properties.      It   is   a   substance   that  it  is 
exceedingly  difficult  to  keep  in  use  for  any  length  of  time. 
It   is   extremely   volatile,   and   in  the  form   of   an   aqueous 
solution  becomes  partially  reduced  with  great  readiness  in 
presence  of  the   slightest   contaminating  particle  of  organic 
matter.       It  is  generally  believed  that  the  aqueous  solutions 
are   reduced  by  light  alone,  but  this  is  not  the  case  :  they 
may  be  exposed  to  the  light  with  impunity  if  dust  be  abso- 
lutely denied  access  to  them.       It  would  even  seem  that  the 
solutions  are   improved  for  some    purposes  by    exposure  to 
sunlight  (vide  infra,  §336,  the  remarks  on  solutions  of  metallic 
salts).      (Some   observations   communicated   to    me    by   Dr. 
Lindsay  Johnson  go  to  prove  that,  if  dust  be  avoided,  solu- 
tions keep  better  in  the  light,  with   occasional   "  sunning," 
than  in  the  dark.) 

Great  stress  is  laid  by  authors  on  the  fact  that  the  vapour  of  osmium  is 
very  irritating  to  mucous  tissues.  It  is  said  that  the  slightest  exposure  to 
it  is  sufficient  to  give  rise  to  serious  catarrh,  irritation  of  the  bronchial  tubes, 
laryngeal  catarrh,  conjunctivitis,  etc.  I  have  never  myself  suffered  in  this 
way,  but  there  is  no  doubt  that  many  persons  do,  and  such  susceptible  sub- 
jects should  be  very  careful  in  handling  osmium  in  any  form. 

34.  Kow  to  keep  the  Solutions. — After  having  carefully  tried 
several  of  the  plans  that  have  been  recommended  for  keep- 
ing the  working  solutions  free   from  dust,   I  have  come  to 
the  conclusion  that  the  following  is  the  most  practical  plan 
for  preventing  them  from  "  going  bad  :  " — The  solution  of 
osmic  acid   in    chromic    acid    solution    is   not,  like  the  solu- 
tion in  pure  water,  easily  reducible,  but  may  be  kept  with- 
out any  special  precautions.      I  therefore  keep  the  bulk  of 


32  CHAPTER    IV. 

my  osmium  in  the  shape  of  a  2  per  cent,  solution  of  osmic 
acid  in  1  per  cent,  aqueous  chromic  acid  solution.  This 
solution  serves  for  fixation  by  osmium  vapours,  and  for 
making  up  solution  of  Flemming,  which  is  the  form  in  which 
osmium  is  most  generally  employed.  A  small  quantity  of 
osmic  acid  may  also  be  made  up  in  1  per  cent,  solution  in 
distilled  water,  and  kept  carefully  protected  from  dust  for 
use  in  special  cases.  Those  who  have  to  do  a  great  deal  of 
fixing  by  means  of  the  vapours  may  also  keep  a  supply  of 
the  solid  oxide  for  this  purpose. 

GRUBLEE  AND  HOLLBOEN  now  send  out  osmic  acid  in  tubes 
containing  one  tenth  of  a  gramme. 

COEI  (Zeit.  f.  wiss.  Mik.}  vi,  4,  1890,  p.  442)  finds  that 
solutions  in  distilled  water  keep  perfectly  if  there  be  added 
to  them  enough  permanganate  of  potassium  to  give  a  very 
slight  rosy  tint  to  the  liquid.  From  time  to  time,  as  the 
solution  becomes  colourless,  further  small  quantities  of  the 
salt  should  be  added,  so  as  to  keep  up  the  rosy  tint. 

35.  Regeneration  of  Reduced  Solutions. — BRISTOL  (Amer. 
Nat.,  xxvii,  1893,  p.  175;  Journ.  Roy.  Mic.  Soc.,  1893,  p. 
564;  Pef.  Handbook  Ned.  Sci.,  Supp.,  p.  442)  says  that 
reduced  solutions  may  be  regenerated  by  oxidising  them  by 
means  of  peroxide  of  hydrogen.  The  reaction  is  stated  to 
be  identical  with  that  which  takes  place  in  the  bleaching  of 
osmium-blackened  tissues  by  peroxide.  It  is  admitted  that 
the  tetroxide  of  osmium,  Os04,  is  reduced  by  contact  with 
organic  matter  into  the  deutoxide,  Os03.  Then  Os 00  -r 
2H303  =  Os04  +  2H3O. 

According  to  Bristol,  for  regenerating  100  c.c.  of  1  per 
cent,  solution  of  osmic  acid  (erratim  10  per  cent,  in  Journ. 
Roy.  Mic.  /Soc.),  ten  to  twenty  drops  of  fresh  peroxide  solu- 
tion should  be  added. 

KOLOSSOW  (Zeit.  f.  wiss.  Mik.,  ix,  1,  1892,  p.  40)  says  that 
half -reduced  solutions,  so  long  as  they  have  not  lost  their 
characteristic  odour,  may  be  clarified  by  the  addition  of  a 
little  powdered  potash -alum. 

But  this  is  evidently  only  a  process  of  clarification,  not 
of  regeneration  ;  the  alum  acting  mechanically  by  carrying 
down  the  suspended  matter,  as  isinglass  does  in  the  "fining  " 


FIXING    AND    HARDENING  AGENTS.  33 

of  beer.      MAYER   finds   that  addition    of  common   salt  will 
produce  the  same  effect. 

36.  Fixation  by  the    Vapours. — Osmic   acid    is    frequently 
employed  in  the  form  of  vapour,  and  its  employment  in  this 
form  is  indicated  in  most  of  the  cases  in  which  it  is  possible 
to  expose  the  tissues  (jirectly  to   the   action  of  the  vapour. 
The   tissues  are   pinned  out  on  a  cork,  which  must  fit  well 
into    a   wide-mouthed   bottle   in   which  is  contained  a  little 
solid  osmic  acid  (or  a  small  quantity  of  1  per  cent,  solution 
will   do).      Very  small   objects,    such   as   isolated   cells,   are 
simply  placed  on  a  slide,  which  is  inverted  over  the  mouth 
of  the  bottle.      They  remain  there  until  they  begin  to  turn 
brown  (isolated  cells  will  generally  be  found  to  be  sufficiently 
fixed  in  thirty  seconds ;  whilst  in   order  to   fix   the  deeper 
layers  of  relatively  thick  objects,  such  as  retina,  an  exposure 
of  several  hours  may  be  desirable).      It  is  well  to  wash   the 
objects  with  water  before  staining,  but  a  very  slight  washing 
will    suffice.       For   staining,   methyl-green  may   be   recom- 
mended for  objects  destined  for  study  in  an  aqueous  medium, 
and,    for    permanent     preparations,     alum-carmine,    picro- 
carmine,  or  haematoxylin. 

In  researches  on  nuclei,  it  is  possible  and  may  be  useful 
to  employ  the  vapours  of  a  freshly  prepared  mixture  of 
osmic  and  formic  or  acetic  acid  (Gilson,  La  Cellule,  i,  1885, 
p.  96). 

The  reasons  for  preferring  the  process  of  fixation  by  vapour  of  osmium, 
where  practicable,  are  that  osmic  acid  is  more  highly  penetrating  when  em- 
ployed in  this  shape  than  when  employed  in  solution,  and  produces  a  more 
equal  fixation,  and  that  the  arduous  washing  out  required  by  the  solutions  is 
here  done  away  with.  lu  many  cases  delicate  structures  are  better  pre- 
served, all  possibility  of  deformation  through  osmosis  being  here  eliminated. 

37.  Fixation  by  Solution. — Osmic  acid  is  now  very  seldom 
used  pure  in  the  shape  of  solutions,  as  it  has  been  found  to 
give  better  results  when  combined  with  other  ingredients,  as 
in  the  mixture  of  FLEMMING.     When,  however,  it  is  employed 
in  pure  aqueous  solutions  it  is  used  in  strengths  varying  from 
-^j  per  cent,  to  1  per  cent.      I  should  say  myself  that,  as  a 
rule,   not  more  than  0*1  per  cent.,  and  never  more  than  0'5 
per  cent.,  should  be  used. 

3 


34  CHAPTEll    IV. 

A  little  acetic  or  formic  acid  (0'5  to  1  per  cent.)  may 
generally  with  advantage  be  added  to  the  solutions  just 
before  using. 

If  solutions  made  with  pure  water  be  used,  they  must  be 
kept  protected  from  the  light  during  the  immersion  of  tissues. 
This  precaution  is  not  necessary  if  Flemming's  or  Hermann's 
solution  be  used.  If  the  immersion  is  to  be  a  long  one  the 
tissues  must  be  placed  with  the  solution  in  well- closed  vessels, 
as  osmium  is  very  volatile. 

38.  After-treatment. — The  excess  of  osmic  acid  must  be  well 
washed  out  before  proceeding  to  any  further  steps  in  prepara- 
tion ;  water  should  be  used  for  washing.  Notwithstanding 
the  greatest  care  in  soaking,  it  frequently  happens  that  some 
of  the  acid  remains  in  the  tissues,  and  causes  them  to  over- 
blacken  in  time,  and  in  any  case  hinders  staining.  To 
obviate  this  it  is  necessary  to  wash  them  out  in  ammonia- 
carmine  or  picro-carmine,  or  to  soak  them  for  twenty-four 
hours  in  a  solution  of  bichromate  of  potash  (Miiller's  solution 
or  Erlicki's  will  do),  or  in  0'5  per  cent,  solution  of  chromic 
acid,  or  in  Merkel's  solution.  The  treatment  with  bichromate 
solutions  has  the  great  advantage  of  highly  facilitating 
staining  with  carmine  or  hsematoxyiin.  Max  Schultze 
recommended  washing,  and  mounting  permanently  in  acetate 
of  potash  •  but  I  believe  the  virtues  attributed  to  this  method 
are  illusory.  Fol  has  recommended  treatment  with  a  weak 
solution  of  carbonate  of  ammonia.  But  the  best  plan  of  all 
is  to  properly  bleach  the  preparations  (see  "Bleaching"). 
This  may  be  done  (as  recommended  by  FOL,  BRASS,  and 
OVERTON)  by  means  of  peroxide  of  hydrogen,  which  regenerates 
the  osmium  to  osmic  acid.  OVERTON  (Zeit.  f.  wiss.  Mik.,  vii, 
•1,  1890)  finds  that  bleaching  is  completed  in  a  few  minutes 
in  a  mixture  of  1  part  commercial  peroxide  of  hydrogen 
with  10  to  25  parts  70  per  cent,  alcohol.  (The  commercial 
peroxide,  slightly  acidulated  with  HC1,  will  keep  well  in  the 
dark  ;  but  the  mixture  with  alcohol  must  be  made  fresh  for 
use.)  CARAZZI'S  peroxide  of  sodium  may  be  found  con- 
venient for  this  purpose.  BINET  (Journ.  de  I'Aiiat.  et  de  la 
PhysioL,  xxx,  1894,  p.  449)  has  successfully  used  perman- 
ganate of  potash.  MONCKEBERG  and  BETHE  (Arch.  mik. 
Anat.,  liv,  1899,  p.  135;  Zeit.  f.  wiss.  Mik.,  xvi,  2,  p.  244) 


FIXING   AND    HARDENING   AGENTS.  35 

have  succeeded  in  satisfactorily  restoring  the  staining  sus- 
ceptibility of  osmium  material  by  means  of  sulphurous  acid 
(obtained  by  adding  hydrochloric  acid  to  bisulphide  of 
sodium,  2  to  4  drops  of  the  acid  added  to  10  c.c.  of  a  2  per 
cent,  solution  of  the  salt).  But  perhaps  the  most  convenient 
method  is  the  original  chlorate  of  potash  method  of  MAYER, 
for  which  see  under  "  Bleaching." 

FOL  (Lefor6.,p.  174)  recommends  a  weak  aqueous  solution  of  ferricyanide 
of  potassium.  MAYER  (Grundzilge,  p.  27)  notes  hereon  that  he  has  had 
tolerable  results  with  it,  though  not  with  the  ferrocyanide,  and  objects  that 
the  ferricyanide  only  acts  in  aqueous  solution,  not  in  alcoholic.  He  objects 
to  peroxide  of  hydrogen  the  instability  of  its  solution,  and  adds  that  the 
peroxide  of  sodium  has  other  disadvantages. 

The  same  stains  recommended  for  objects  fixed  by  the  vapours  will  be 
found  useful  here.  For  sections,  of  course,  in  both  cases  safranin  and  other 
anilin  stains  may  be  employed  with  advantage,  as  may  haematoxylin. 

39.  Characters  of  the  Fixation  with  Osmic  Acid. — In  general 
osmic  acid,  especially  when  used  in  the  form  of  vapour,  fixes 
protoplasm  faithfully,  nuclei  badly,  and  there  are  other 
drawbacks  over  and  above  those  before  mentioned  (§  27). 
The  penetrating  power  of  the  solutions  is  very  low,  so  that  if 
any  but  very  small  pieces  of  tissue  be  taken  the  outer  layers 
become  over-fixed  before  the  action  of  the  reagent  has  pene- 
trated to  the  deeper  layers.  Over-fixed  cells  have  a  certain 
homogeneous,  glassy,  or  colloid  look,  owing  to  all  their  con- 
stituents having  been  raised  by  coagulation  to  so  high  an 
index  of  refraction  that  little  or  no  detail  is  visible  in  them. 
They  stain  very  badly,  or  not  at  all.  Such  cells  are  known 
as  "  osmicated  cells,  oxmirte  Zellen."  There  is  no  remedy 
for  this  state  of  things  if  once  it  has  occurred.  For  this 
reason  it  is  important  to  avoid  using  stronger  solutions  than 
is  necessary.  The  danger  of  osmication  is  lessened  by  using 
the  osmic  acid  in  conjunction  with  certain  other  reagents, 
such  as  chromic  acid.  But  it  is  not  thereby  entirely 
removed ;  FLEMMING'S  mixture,  especially  the  strong  formula, 
will  readily  osmicate  superficial  cells  if  care  be  not  taken. 
For  ordinary  histological  work  osmication  of  superficial  layers 
is  not  of  much  consequence.  But  for  cytological  work  care 
should  be  taken  not  to  draw  conclusions  as  to  the  structure 
of  cells  from  osmicated  specimens,  and  attention  should  be 
confined  to  cells  four  or  five  layers  deeper  down,  which  will 


36  CHAPTER  IV. 

generally   be    found    to    present    the    desired    intensity    of 
fixation. 

Osmic  acid  stains  certain  fatty  bodies  black;  it  should  therefore  be 
avoided  for  tissues  in  which  much  fat  is  present ;  or  if  not,  the  preparations 
should  be  subsequently  very  thoroughly  bleached,  or  the  blackened  fat 
may  be  afterwards  dissolved  out  by  means  of  oil  of  turpentine.  See  Fat. 

According  to  ALTMANN,  STAEKE,  and  HANDWEECK,  only  free  oleic  acid 
and  olein  are  directly  blackened  by  osmic  acid,  stearin  and  palmitin,  and 
stearic  and  palmitic  acid  are  only  browned  by  it,  with  an  after-blackening 
which  is  produced  by  subsequent  treatment  with  alcohol.  Neither  reaction 
occurs  with  the  fatty  bodies  in  the  solid  state,  and  can  only  be  obtained  when 
they  are  either  in  a  state  of  fusion  or  solution  (from  the  paper  of  HAND- 
WEECK in  Zeit.f.  wiss.  Mik.,  xv,  2,  1898,  p.  177). 

39a.  Osmic  Mixtures. — The  chief  osmic  mixtures  are  those 
of  FLEMMING  and  of  HERMANN,  for  which  see  §§  46,  47,  and 
50.  The  following,  however,  may  be  mentioned  here. 

KANVIEE  ET  VIGNAL  (RANVIEB,  Leg.  d'Anat.  Gen.,  "  App.  term,  des 
muscles  de  la  vie  org.,"  p.  76  ;  VIGNAL,  Arch,  de  Physiol.,  1884,  p.  181) 
take  equal  volumes  of  1  per  cent,  osmic  acid  and  90  per  cent,  alcohol  (freshly 
mixed).  They  wash  out  in  80  per  cent,  alcohol,  then  wash  with  water  and 
stain  for  forty-eight  hours  in  picro-carrnine  or  hsematoxylin.  Viallanes  has 
applied  this  method  to  the  histology  of  insects. 

KOLOSSOW  (Zeit.  f.  wiss.  Mik.,  v,  1,  1888,  p.  51)  has  recommended  a  0'5 
per  cent,  solution  of  osmic  acid  in  2  or  3  per  cent,  solution  of  nitrate  or 
acetate  of  uranium,  as  having  a  greatly  enhanced  penetrating  power. 

He  has  more  lately  (op.  cit.,  ix,  1,  1892,  p.  39)  recommended  for  the  same 
reason  a  mixture  of  50  c.c.  absolute  alcohol,  50  c.c.  distilled  water,  2  c.c. 
concentrated  nitric  acid,  and  1  to  2  grin,  osmic  acid.  This  mixture  is  said  to 
keep  indefinitely  in  a  cool  place. 

BUSCH  (Neurol.  Centralb.,  xvii,  1898,  No.  10,  p.  476  ;  Zeit.  f.  wiss.  Mik., 
xv,  3,  p.  373)  holds  that  the  penetration  of  osmic  acid  is  enhanced  by  com- 
bining it  with  iodate  of  sodium,  which  by  hindering  its  too  rapid  decompo- 
sition in  the  tissues  ensures  a  more  energetic  action  in  the  deeper  layers. 
He  adds.3  per  cent,  of  sodium  iodate  to  a  l,per  cent,  solution  of  osmic  acid. 

This  mixture  appears  to  me  rational,  whilst  the  above-mentioned  mixtures 
with  alcohol  do  not.  Alcohol  is  a  reducing  agent,  and  therefore  surely  in- 
compatible with  so  easily  reducible  a  substance  as  osmic  acid,  which  should 
rather  be  combined  with  oxidising  agents.  Now  sodium  iodate  is  a  power- 
ful oxidising  agent,  and  so  far  seems  quite  indicated. 

40.  Chromic  Acid. — Chromic  anhydride,  Cr03,  is  found  in 
commerce  in  the  form  of  red  crystals  that  dissolve  readily  in 
water,  forming  chromic  acid,  H2Cr04.  These  crystals  are 
very  deliquescent,  and  it  is  therefore  well  to  keep  the  acid 
in  stock  in  the  shape  of  a  1  per  cent,  solution.  Care  must 


FIXING    AND    HARDENING    AGENTS.  37 

be  taken  not  to  allow  the  crystals  to  be  contaminated  by 
organic  matter,  in  the  presence  of  which  the  anhydride  is 
readily  reduced  into  sesquioxide. 

Chromic  acid  is  generally  employed  in  aqueous  solution. 
Some  observers  (KLEIN  ;  URBAN  PRITCHARD  ;  PERENYI)  have 
recommended  alcoholic  solutions ;  but  this  would  appear  to 
be  an  irrational  practice.  For  in  the  presence  of  alcohol 
chromic  acid  has  a  great  tendency  to  become  reduced  to 
chromous  oxide  or  sesquioxide,  neither  of  which  appears  to 
have  any  fixing  power. 

The  most  useful  strengths  in  which  it  is  employed  in  aqueous 
solution  are  from  0*1  to  1*0  per  cent,  for  a  period  of  immersion 
of  a  few  hours  (structure  of  cells  and  ova) .  For  nerve  tissues  :, 
weaker  solutions  are  taken,  -5^y  to  •§-  per  cent,  for  a  few 
hours.  Stronger  solutions,  such  as  5  per  cent.,  should  only 
be  allowed  to  act  for  a  few  seconds. 

The  objects  should  be  washed  out  with  water  before 
passing  into  alcohol  or  staining  fluids.  Long  washing  in 
water  is  necessary  to  prepare  them  for  staining,  except  an 
anilin  stain  be  used.  It  is  possible  to  wash  out  in  alcohol, 
and  this  may  be  useful  in  special  cases,  but  in  general  I 
think  the  practice  is  not  to  be  recommended.  It  is  well  to 
wash  for  many  hours  in  running  water. 

Mayer  notes  her  eon  (Grundzuge)  that  the  operation  of 
washing  may  be  done  away  with  in  the  following  manner  : — 
The  fixed  material  is  merely  rinsed  in  water  and  brought 
direct  into  70  per  cent,  alcohol.  It  is  washed  therein, 
preferably  in  the  dark  (see  §  41),  until  after  several  changes 
the  alcohol  remains  colourless.  It  is  then  either  passed 
through  higher  alcohols  and  imbedded  in  paraffin,  the 
chromous  oxide  (or  whatever  chrome  compound  it  may  be 
that  is  present  in  the  tissues)  being  removed  from  the 
sections  after  these  are  made ;  or  this  necessary  removal  is 
performed  at  once.  If  this  be  preferred,  the  material  is 
brought  into  sulphuric  acid  diluted  with  twenty  volumes  of 
water,  or  into  nitric  acid  diluted  with  ten  volumes  of  water. 
After  at  most  a  few  hours  therein,  it  will  have  become  of  a 
light  greyish  green,  and  on  removal  of  the  acid  may  be 
readily  stained.  If  it  be  preferred  to  treat  the  sections,  it  is 
sufficient  to  put  them  into  the  usual  hydrochloric  acid  alcohol 
(four  to  six  drops  of  HC1  to  100  c.c.  of  70  per  cent,  alcohol), 


38  CHAPTER    IV. 

in  which  after  a  short  time  they  become  almost  white,  and 
will  stain  excellently  with  any  of  the  usual  stains.  Unna 
(Arch.  f.  mik.  Anat.,  xxx,  1887,  p.  47,  see  Journ.  R.  Mic. 
8oc.,  1887,  p.  1060)  holds  that  the  chrome  is  present  in  the 
tissues  in  the  form  of  chromic  chromate,  and  removes  it  by 
treatment  with  peroxide  of  hydrogen.  Overton  (Zeit.  f. 
wiss.  Mik.,  vii,  1890,  p.  9,  employs  a  weak  solution  of  sul- 
phurous acid,  which  converts  it  into  a  sulphate.  See  also 
the  directions  for  bleaching  osmic  acid  preparations,  §  38. 

Tissues  that  have  been  fixed  in  chromic  acid  are  usually 
stained  in  aqueous  solutions,  as  it  is  held  that  water  does 
not  have  an  injurious  effect  on  them ;  the  acid  entering  into 
some  chemical  combination  with  the  elements  of  the  tissues, 
forming  with  them  a  compound  that  is  not  affected  either 
physically  or  chemically  by  water.  But  there  is  reason 
to  doubt  whether  the  hereby  alleged  insolubility  of  the 
elements  is  as  thoroughgoing  as  is  generally  believed ;  see, 
for  instance,  the  paper  of  TULLYESNICZKY,  Arch.f.  mik.  Anat., 
lii,  1898,  p.  221. 

The  best  stain  for  chromic  material  that  has  not  been 
treated  by  Mayer's  special  process,  or  by  a  similar  one,  is 
hsematoxylin,  or,  for  sections,  some  anilin  stain.  But  the 
previous  washing  out  with  water  must  be  very  thorough  if 
good  results  are  to  be  insured  ;  it  may  take  days. 

Chromic  acid  is  not  a  very  penetrating  reagent,  and  for 
this  reason,  as  well  as  for  others,  is  now  seldom  used  pure 
for  fixing,  but  plays  an  important  part  in  the  mixtures 
described  below,  of  which  the  chief  is  certainly  the  mixture 
of  Flemming.  - 

For  prolonged  hardening  chromic  acid  is  generally  employed 
in  strengths  of  4-  per  cent,  to  |  per  cent.,  the  immersion 
lasting  a  few  days  or  a  few  weeks,  according  to  the  size  and 
nature  of  the  object.  Mucous  membrane,  for  instance,  will 
harden  satisfactorily  in  a  few  days ;  brain  will  require  some 
six  weeks. 

Large  quantities  of  the  solution  must  be  taken  (at  least 
200  grammes  for  a  piece  of  tissue  of  1  centimetre  cube — 
Ranvier) . 

In  order  to  obtain  the  best  results  you  should  not  employ 
portions  of  tissue  of  more  than  an  inch  cube.  For  a  human 
spinal  cord  you  should  take  two  litres  of  solution,  and  change 


FIXING    AND    HARDENING    AGKNTS.  39 

it  for  fresh  after  a  few  days.-  Six  weeks  or  two  months  are 
necessary  to  complete  the  hardening. 

The  solution  should  be  taken  weak  at  first,  and  the  strength 
increased  after  a  time.  The  objects  should  be  removed  from 
the  solution  as  soon  as  they  have  acquired  the  desired  con- 
sistency, as  if  left  too  long  they  will  become  brittle.  (These 
precautions  are  peculiarly  necessary  in  the  case  of  chromic 
acid.)  They  may  be  preserved  till  wanted  in  alcohol  (95  per 
cent.).  It  is  well  to  wash  them  out  in  water  for  twenty-four 
or  forty-eight  hours  before  putting  them  into  the  alcohol.  I 
think  it  is  frequently  useful  to  add  a  little  glycerin  to  the 
hardening  solution  ;  there  is  less  brittleness  and,  I  think,  less 
shrinkage. 

Chromic  acid  is  a  most  powerful  and  rapid  hardening 
agent.  (By  it  you  may  obtain  in  a  few  days  a  degree  of 
hardening  that  you  would  hardly  obtain  in  as  many  weeks 
with  bichromate,  for  instance.)  It  has  the  defect,  of  a  great 
tendency  to  cause  brittleness. 

41.  Action  of  lighten  alcohol  containing  chromic  objects. — When  objects 
that  have  been  treated  by  chromic  acid  or  a  chromate  are  put  into  alcohol 
for  hardening  or  preservation,  it  is  found  that  after  a  short  time  a  fine  pre- 
cipitate is  thrown  down  on  the  surface  of  the  preparations,  thus  forming  a 
certain  obstacle  to  the  further  penetration  of  the  alcohol.     Previous  washing 
by  water  does  not  prevent  the  formation  of  this  precipitate,  and  changing 
the  alcohol  does  not  prevent  it  from  forming  again  and  again.     It  has  been 
found  by  Hans  Virchow  (Arch.f.  mik.  Anat.,  Bd.  xxiv,  1885,  p.  117)  that 
the  formation  of  this  precipitate  may  be  entirely  prevented   by  simply 
keeping  the  preparations  in  the  dark.     The  alcohol  becomes  yellow  as  usual 
(and  should  be  changed  as  often  as  this  takes  place),  but  no  precipitate  is 
formed.     If  this  precaution  be  taken,  previous  washing  with  water  may  be 
omitted,  or  at  all  events  greatly  abridged. 

42.  Chromic  Acid  and  Spirit  (URBAN  PBITCHABD,  Quart.  Journ.  Mic. 
Sci.,  1873,  p.  427). — Chromic  acid,  1  part ;  water,  20  parts  ;  rectified  spirit, 
180  parts.     Dissolve  the  chromic  acid  in  the  water  first,  and  then  add  the 
spirit  (violent  action  will  ensue  if  the  dry  chromic  acid  be  added  directly  to 
the  spirit).     The  colour  of  the  solution  soon  becomes  brown.     If  after  a 
few  days  it  turns  semi-gelatinous,  it  should  be  changed  for  fresh.     From  a 
week  to  ten  days  is  required  to  harden  such  tissues  as  retina,  cochlea,  etc., 
for  which  this  fluid  used  to  be  considered  particularly  well  adapted. 

A  mixture  of  2  parts  of  ~  per  cent,  chromic  acid  solution  with  one  part  of 
methylated  spirit  was  much  used  by  KLEIX  in  his  investigations  into  the 
structure  of  cells  and  nuclei,  and  found  to  give  better  results  than  the 
ordinary  reagents  (including  even  osmic  acid). 

Both  these  mixtures  are  seemingly  irrational  (see  §  40).    MAYEE  (Grund- 


40  CHAPTER    IV. 

zilge)  remarks  on  Pritchard's  formula — -"An  altogether  silly  receipt;  after 
a  short  time  the  mixture  has  lost  its  acid  reaction,  and  then  only  the 
alcohol  can  act."  See  also  the  remarks  on  the  mixture  of  PEBENYI,  §  52. 

43.  Chromo-acetic  Acid  (FLEMMING,  Zellslz.,  Kern  u.  ZelUh., 
p.  382). 

Chromic  acid  .      0'2  to  0'25  per  cent. 

Acetic  acid   .  .0*1  per  cent.,  in  water. 

Flemming  found  this  the  best  reagent  for  the  study  of 
the  achromatic  elements  of  karyokinesis.  (Flemming  wrote 
this  in  1882,  and  I  doubt  whether  it  would  now  hold  good.) 
Stain  with  hsematoxylm  (the  preparations  are  not  favourable 
for  staining  with  safranin  or  other  coal-tar  colours). 

The  following  has  been  recommended  as  a  good  fixing  and 
hardening  mixture  for  Annelids  in  general,  and  probably  for 
other  forms,  by  EHLEES  (I  do  not  know  whether  it  has  been 
published  elsewhere) : — To  100  c.c.  of  chromic  acid  of  0'5  to 
1  per  cent,  add  from  1  to  5  drops  of  glacial  acetic  acid.  The 
proportion  of  acetic  acid  indicated  is  said  to  be  sufficient  to 
counteract  any  tendency  to  shrinkage  due  to  the  chromic 
acid. 

Similar  to  this  is  the  C(  chromo-acetic  acid,  No.  1,"  of  Lo 
BIANCO  (Mitth.  Zool.  Stat.  Neapel,  ix,  1890,  p.  443),  viz.  1 
part  50  per  cent,  acetic  acid  and  20  parts  1  per  cent,  chromic 
acid,  which  is  found  very  useful  for  fixing  marine  animals. 

44.  Chromo-formic   Acid  (RABL,    Morph.    Jahrb.,    x,    1884, 
pp.  215,  216). — Four  or  five  drops  of    concentrated   formic 
acid  are  added  to  '200  c.c.  of   0*33  per    cent,  chromic  acid 
solution.       The    mixture   must   be    freshly   prepared   at   the 
instant  of  using.     Fix  for  twelve  to  twenty-four  hours,  wash 
out  with  water.     Used  by  Eabl  for  the  study  of  karyokinesis. 

45.  Chromo-osmic  Acid  (MAX  FLESCH,  Arch.f.  mik.  Anat,xv},1879, 
p.  300).— This  mixture  (osmic  acid  O'lO,  chromic  acid  0'25,  water  lOO'O) 
may  for  almost  all  purposes  be  considered  to  be  superseded  by  that  of 
Flemming,  §  46. 

Lo  BIANCO  (Mitth.  Zool.  Stat.  Neapel,  ix,  1890,  p.  443) 
employs  for  marine  animals  a  mixture  of  1  part  1  per  cent, 
osmic  acid  and  50  parts  1  per  cent,  chromic  acid. 

46.  Chromo-aceto-osmic  Acid  (FLEMMING,  Zellsubstanz,  Kern 
~und  Zelltheilung,  1882,  p.  381),  FIEST  or  WEAK  formula  : 


FIXING  AND    HARDENING  AGENTS.  41 

Chromic  acid       .  .      0*25  per  cent.^j 

Osmic  acid  .  .0*1     per  cent,  j-  In  water. 

Glacial  acetic  acid  .  0*1  per  cent.J 
This  liquid  may,  without  inconvenience,  be  allowed  to  act 
for  many  hours  or  days,  or  according  to  some  workers  even 
weeks  or  months.  Wash  out  very  thoroughly  in  water  (see 
also  §  40).  Stain  with  haematoxylin  if  you  wish  to  stain  in 
toto  (staining  in  this  way  with  other  reagents  is  possible,  but 
very  difficult,  and  not  to  be  recommended).  Stain  sections 
with  safranin  or  other  coal-tar  colour,  or  with  hasmatoxylin 
or  Kernschwarz. 

To  make  up  this  mixture  with  the  usual  stock  solutions, 
you  take — 

Chromic  acid  of  1  per  cent.    .  .      25  volumes. 

Osmic  acid  of  1  per  cent.        .  .10        ,, 

Acetic  acid  of  1  per  cent.       .  .10        „ 

Water          .  .  .  .  55        ,, 

If  you  keep  your  osmium  in  2  per  cent,  solution  in 
chromic  acid  of  1  per  cent.,  as  I  have  recommended,  you 
will  have  to  take  only  20  vols.  of  chromic  acid,  5  of  your 
osmium  solution,  and  65  of  water.  See  also  the  remarks  on 
the  deterioration  of  these  solutions  by  keeping,  in  the  next 
section. 

It  is  not  necessary  in  all  cases  to  observe  the  exact  proportions  of  the  in- 
gredients in  this  mixture.  FOL  (Lehrb.  d.  vergl.  mik.  Anat.,  1884,  p.  100) 
recommends  the  following : 

1  per  cent,  chromic  acid 25  vols. 

1  per  cent,  osmic  acid        .         .         .         .         .       2    „ 

2  per  cent,  acetic  acid 5    „ 

Water 68    „ 

— that  is  to  say,  a  mixture  much  weaker  in  osmium  than  Flemming's. 

A  mixture  still  weaker  than  this  is  osmium,  viz.  with  1  vol.  osmic  acid 
solution  instead  of  2,  has  been  recommended  by  GOBI  (Zeit.f.  wiss.  Mik.» 
vi,  1,  1890,  p.  441). 

This  mixture,  though  less  in  vogue  now  than  the  follow- 
ing or  strong  mixture,  is  one  of  the  most  celebrated  of  fixa- 
tives. I  think  justly  so,  for,  with  the  possible  exception  of 
HERMANN'S  mixture,  no  known  fixative  seems  to  me  to  afford 
such  fine  images  of  cellular  structures  as  Flemming's  two 
fluids.  That  is  to  say,  that  the  fixation  is  pre-eminent  both 
as  regards  the  preservation  of  the  structures  and  as  regards 
their  optical  differentiation.  But  this  is  meant  with  the  re- 


42  CHAPTER    IV. 

servation  that  the  reagent  must  be  properly  used,  and  not 
applied  to  objects  for  which  it  is  not  fitted.  For  instance, 
its  power  of  penetration  is  extremely  bad ;  it  will  not  fix 
properly,  even  in  a  loose-celled  tissue,  through  more  than  a 
layer  of  about  five  cells  thick.  It  is  therefore  suitable  only 
for  very  small  objects  or  for  very  small  pieces  of  tissue,  such 
as  suffice  for  cytological  or  histological  work.  But  it  is  not 
suitable  at  all  for  voluminous  objects,  such  as  the  organo- 
logical  anatomist  and  the  embryologist  so  frequently  have  to 
do  with.  It  has  not  the  character  of  a  general  reagent. 

In  previous  editions  I  have  quoted  some  authors  who  have 
accused  this  reagent  of  faulty  preservation.  It  must  be 
admitted  that  it  has  the  defect  of  easily  causing  over-fixation 
of  superficial  cells,  and,  owing  to  its  defective  penetration, 
insufficient  fixation  of  deep-lying  ones.  It  requires  to  be 
used  in  the  proper  manner,  and,  above  all,  with  the  proper 
objects  ;  and  it  is  by  no  means  of  such  universal  applicability 
as  has  been  supposed. 

It  may  be  used  for  prolonged  hardening,  e.  g.  of  nervous 
tissue,  and  is  a  very  good  reagent  for  the  purpose. 

47.  Chromo-aceto-osmic  Acid  (FLEMMING,  Zeit.  f.  iciss.  Mik., 
1,  1884,  p.  349),  SECOND  or  STRONG  formula  : 

1  per  cent,  chromic  acid     .  .15  parts. 

2  per  cent,  osmic  acid         .  .        4       ,, 
Glacial  acetic  acid      ...        1  part. 

If  2  per  cent,  osmic  acid  solution  should  not  be  at  hand,  you  may  conve- 
niently make  the  mixture  by  taking — 

10  per  cent,  chromic  acid         .         .         .         .15  parts. 
1  per  cent,  osmic  acid      .         .         .         .         .     80     ,, 
Glacial  acetic  acid  .         .         .         .        .'       .     10     „ 
Water 95     „ 

If  this  mixture  be  kept  in  stock  in  large  quantities,  it 
may  go  bad,  probably  on  account  of  the  large  proportion  of 
organic  acid  contained  in  it.  I  therefore  recommend  that  it 
be  made  up  from  time  to  time  from  stock  solutions,  in  which 
the  osmic  acid  is  kept  separate  from  the  acetic  acid;  the 
proportions  being  as  follows  : 

CrOs 0-15 

Os04    ....  .       0-08 

Acid.  acet.     .  .  .  .  .1*00 

Aq '  19-00 


FIXING    AND    HARDENING    AGENTS.  43 

You  may  make  up  and  keep  separately — 

(A)   1  per  cent,  chromic  acid  .  .11  parts, 

Distilled  water         .  .  4       „ 

Glacial  acetic  acid  ...  1  part, 
and  (B)  a  2  per  cent,  solution  of  osmic  acid  in  1  per  cent, 
chromic  acid  solution,  and  when  required,  mix  four  parts  of 
A  with  one  of  B ;  or,  of  course,  if  you  prefer  it,  you  may 
keep  the  osmic  and  chromic  acid  ready  mixed  in  the  propor- 
tions given,  and  add  5  per  cent,  of  acetic  acid  at  the  moment 
of  using. 

According  to  Flemming,  it  is  better  not  to  make  up  very 
large  quantities  of  the  mixture  at  once,  as  osmium  being 
very  volatile  it  will  be  found  that  solutions  that  have  been 
long  in  use  no  longer  contain  the  proper  proportion  of  that 
ingredient,  and  the  hardening  action  being  thus  weakened 
the  swelling  action  of  the  acetic  acid  may  be  insufficiently 
controlled. 

Merk  (Denksch.  d.  Math.  Naturw.  Cl.  d.  K.  Acad.  d.  Wiss.  Wien,  1887  ; 
cf.  Zeit.f.  wiss.  Mile.,  v,  2, 1888,  p.  237)  proposes  to  make  up  separately  (A) 
2  per  cent,  chromic  acid          .         .         .         .7*5  parts, 

Water 3'5     „ 

Acetic  acid 1     part, 

and  (B),  some  1  per  cent,  osmic  acid  solution,  and  to  mix  for  use  12  parts  of 
A  with  8  of  B.  But  this  plan  leaves  you  in  the  old  difficulty  of  keeping 
your  osmium  in  aqueous  solution. 

It  does  not  appear  necessary  to  observe  the  exact  proportions  of  the  ingre- 
dients of  these  mixtures,  a  certain  latitude  is  allowable.  Thus  CAENOY  (La 
Cellule,  i,  2,  1885,  p.  211)  has  employed  a  mixture  one  third  stronger  in 
osmic  acid  and  twice  as  strong  in  chromic  acid,  viz. — 

Chromic  acid  of  2  per  cent,  (or  even  stronger)     45  parts. 

Osmic  acid  of  2  per  cent 16     „ 

Glacial  acetic  acid  .         .         .         .         .  3     ,, 

PODWYSSOZKI  recommends  (for  glands  especially)  the  following  modifi- 
cation : 

1  per  cent.  CrO3  dissolved  in  0'5  per  cent,  solution  of 
corrosive  sublimate  .......     15  c.c. 

2  per  cent,  osmic  acid  solution      .....       4  c.c. 
Glacial  acetic  acid         .         .         .         .         .         .  6  to  8  drops. 

The  sublimate  is  said  to  augment  the  penetration  of  the  osmium,  but  is 
unfavourable  to  staining.  The  proportion  of  acetic  acid  is  reduced  in  order 
to  avoid  swelling  of  the  tissue  elements  (ZIEGLEE'S  Seitrdge  z.  path.  Anat., 
\,  1886  ;  cf.  Zeit.f.  wiss.  Mik.t  iii,  3,  1886,  p.  405). 

The  characters  of  the  fixation  are,  in  the  main,  those  of 
the  weak  formula,  but  the  action  is  more  energetic,  the 


44  CHAPTER    IV. 

tissues  are  more  difficult  to  stain,  but  afford,  when  obtained, 
a  more  delicate  and  selective  stain,  especially  showing  up 
karyokinetic  figures  and  nucleoli. 

The  strong  formula  was  recommended  by  FLEMMING  in  the 
first  instance  merely  for  a  very  special  purpose,  the  hunting 
for  karyokinetic  figures,  and  not  for  general  purposes. 
Further  experience  has  shown  that  it  is  applicable  to  other 
cytological  purposes,  and  is  in  many  cases  considerably 
superior  to  the  weak  formula.  But  it  is  not  suited  to  all 
objects,  and  FLEMMING  has  himself  pointed  out  that  some 
workers  have  used  it  for  purposes  for  which  it  is  not  fitted. 
It  is  indeed,  I  think,  even  less  of  a  general  reagent  than  the 
weak  formula,  though  unsurpassed  in  its  own  peculiar 
sphere. 

The  strong  mixture  does  not  brown  tissues  more  than  the 
weak  mixture,  but  rather  less. 

Fat  is  blackened  by  both  these  mixtures.     See  §  39. 

48.  Osmic  Acid  and  Bichromate. — ALTMANN  (Die  Elementarorgan- 
ismen,  Leipzig,  1890;  Zeit.f.  wiss.  Mik.,  vii,  2,  1890,  p.  199)  recommends 
a  mixture  of  equal  parts  of  5  per  cent,  solution  of  bichromate  of  potash 
and  2  per  cent,  solution  of  osmic  acid  for  the  demonstration  of  his  granula. 
The  bichromate,  he  says,  ought  not  to  contain  any  free  chromic  acid,  and  the 
mixture  is  best  prepared  freshly  when  required. 

Lo  BIANCO  (Mitth.  Zool.  Stat.  Neapel,  ix,  1890,  p.  443)  employs  for  marine 
animals  a  mixture  of  100  c.c.of  5  percent,  solution  of  bichromate  and  2  c.c. 
of  1  per  cent,  osmic  acid. 

Evidently,  I  think,  the  addition  of  acetic  acid  to  either  of 
these  mixtures  is  in  most  cases  indicated.  This  step  has,  in 
fact,  been  taken  by  HOE.HL  (Arch.  f.  Anat.  u.  Phys.,  Anat. 
Abth.,  1896,  p.  31 ;  Zeit.  f.  wiss.  Mik.,  xiii,  2,  1896,  p.  227), 
who  recommends  a  mixture  of  80  c.c.  of  3  per  cent,  bichro- 
mate, 20  c.c.  of  1  per  cent,  osmic  acid,  and  2  c.c.  of  glacial 
acetic  acid. 

49.  Osmic,  Bichromate,  and  Platinic  Mixture  (LINDSAY 
JOHNSON'S  Mixture). — Latest  formula,  1895,  communicated 
by  Dr.  Lindsay  Johnson  : 

Bichromate  of  potash  (2*5  per  cent.)  .      70  parts. 

Osmic  acid  (2  per  cent.)     .           .  .      10      ,, 

Platinic  chloride  (1  per  cent.)      .  15      ,, 

Acetic  or  formic  acid  5 


FIXING    AND    HARDENING    AGENTS.  45 

It  is  not  well  to  take  the  platinum  chloride  stronger  than 
here  given,  as  too  strong  solutions  have  a  tendency  to  crys- 
tallise out  on  the  tissues.  HENNEGUY,  who  has  worked  a 
great  deal  with  this  reagent,  and  recommends  it  highly,  says 
(Leqons  sur  la  Cellule,  Paris,  Carre,  1896,  p.  61)  that  it  is 
well  only  to  add  the  acetic  or  formic  acid  just  before  using, 
as  it  frequently  provokes  a  spontaneous  reduction  of  the 
osmium  and  platinum  to  such  an  extent  that  the  mixture  be- 
comes quite  black. 

This  mixture  was  imagined  for  the  preliminary  hardening 
of  retina,  being  allowed  to  act  for  two  hours  only,  and  then 
being  followed  by  final  hardening  in  pure  bichromate  solution. 
But  it  has  proved  applicable  to  other  structures,  and  gives- 
excellent  results.  The  function  of  the  osmic  acid  in  the 
present  formula  is  to  enhance  the  hardening  energy  of  the 
mixture.  Dr.  Lindsay  Johnson  writes  me  that  "  it  greatly 
reduces  the  length  of  time  necessary  for  hardening,  three 
days  being  the  time  from  removal  of  the  organ  to  its  being 
in  celloidin  under  dilute  spirit/' 

This  mixture  may  be  used  for  fixing,  in  some  cases  with 
the  best  results.  HENNEGUY  (/.  c.)  says  it  contracts  the  more 
spongy  sorts  of  protoplasm  less  than  mixture  of  FLEMMING. 
I  think  highly  of  it — for  certain  objects. 

50.  Platino-aceto-osmic  Acid  (HERMANN'S  solution). — This 
valuable  reagent  is  historically  a  modification  of  Flemming's 
solution,  platinum  chloride  being  taken  instead  of  chromic 
acid.  HERMANN  (Arch.  f.  miJc.  Anat.,  xxxiv,  1889,  p.  58) 
substitutes  1  per  cent,  platinic  chloride  for  the  chromic  acid 
in  Flemming's  strong  formula  for  chromo-aceto-osmic  acid 
(§  47),  the  other  ingredients  either  remaining  as  before,  or 
the  osmium  being  diminished  one  half ;  thus,  1  per  cent, 
platinic  chloride  15  parts,  glacial  acetic  acid  1  part,  and  2 
per  cent,  osmic  acid  either  4  parts  or  only  2  parts.  Hermann 
found  that  protoplasmic  structures  are  thus  better  preserved 
than  with  the  chromic  mixture. 

The  after-treatment  and  staining  should  be  the  same  as 
for  objects  treated  with  Flemming's  solution. 

After  considerable  experience  of  this  reagent  I  find  that 
it  has  the  advantage  of  giving  more  colourless  preparations, 
and  it  may  be,  in  some  cases,  a  more  delicate  fixation.  But 


46  CHAPTER    IV. 

the   fixation    is    certainly   not    in    all    cases    superior.      See 
further  under  (i  Ci/tologicaJ  Methods." 

51.  Nitric  Acid  (ALTMANN,  Arch.  Anat.  u.  Phys.,  1881,  p.  219).— 
Altmann  employs  for  fixing  dilute  nitric  acid,  containing  from  3  to  3^  per 
cent,  pure  acid.  Such  a  solution  has  a  sp.  gr.  of:  about  1'02  ;  an  areometer 
may  conveniently  be  used  to  determine  the  concentration  of  the  solution. 
Stronger  solutions  have  been  used,  but  do  not  give  such  good  final  results. 
After  extensive  trial  I  find  ALTMANN'S  solution  to  be  a  good  reagent,  but 
rather  weak. 

His  (ibid.,  1877,  p.  115)  recommended  a  10  per  cent,  solution.  Flemming 
at  one  time  employed  solutions  of  40  to  50  per  cent,  for  the  ova  of  Inverte- 
brates. This  of  course  has  the  advantage  of  a  very  rapid  fixing  action. 

The  action  of  nitric  acid  as  a  fixative  has  been  lately  investigated  by 
TELLYESNICZKY  (Arch.  mik.  Anat,  Hi,  2,  1898,  p.  222).  He  thinks  that 
"for  general  cell-fixing"  the  proper  strength  is  2  per  cent,  to  2^  per  cent., 
as  stronger  grades  act  too  energetically  on  the  superficial  layers.  His  re- 
sults, or  the  interpretation  of  them,  are  therefore  not  quite  concordant  with 
mine. 

Nitric  acid  has  the  valuable  property  of  hardening  yolk  without  making 
it  brittle. 

Pure  water  should  in  no  case  be  used  for  washing  out  after  nitric  acid  ; 
the  preparations  should  be  brought  direct  into  alcohol,  as  recommended  by 
Altmann.  Some  persons  take  absolute  alcohol,  but  I  should  say  70  per  cent, 
is  more  generally  indicated.  Ilabl  has  employed  for  washing  out  a  1  or  2 
per  cent,  solution  of  alum. 

For  prolonged  hardening,  strengths  of  from  3  to  10  per  cent,  are  some- 
times employed.  A  strength  of  12  per  cent.,  allowed  to  act  for  two  or  three 
weeks,  is  said  to  afford  very  tough  preparations  of  the  encephalon. 

BENDA  (Verh.  Anat.  Ges.,  1888;  Ergeb.  d.  Anat.,  i,  1891,  p.  7)  fixes  for 
twenty-four  to  forty-eight  hours  in  10  per  cent,  nitric  acid,  and  then  brings 
the  preparations  direct  into  a  cold  saturated  solution  of  bichromate  of  potash 
diluted  with  three  vols.  of  water.  After  a  few  hours  this  solution  is  changed 
for  a  stronger  one,  and  the  strength  is  gradually  increased  in  such  a  manner 
as  to  arrive  at  a  concentration  of  one  vol.  of  the  saturated  solution  to  one  of 
water  in  two  or  three  days  (or,  for  encephalon  and  spinal  cord  only  in  four- 
teen days).  This  process,  which  is  of  general  applicability  (except  in  so  far 
as  epidermis,  especially  that  of  embryos,  may  be  loosened  by  the  nitric  acid), 
is  said  to  furnish  very  tough  preparations. 

Fol's  Mixture. — Three  vols.  of  nitric  acid,  with  97  vols.  of  70  per  cent. 
alcohol  (verbally  communicated  to  me  by  Prof.  Fol). 

52,  Chromo-nitric  Acid  (PURENYI'S  formula,   Zooi.    Anzeig., 
v,  1882,  p.  459)  : 

4  parts  10  per  cent,  nitric  acid. 

3  parts  alcohol. 

3  parts  0'5  per  cent,  chromic  acid. 


FIXING    AND    HARDENING    AGENTS.  47 

These  are  mixed,  and  after  a  short  time  give  a  fine  violet- 
coloured  solution. 

The  objects  are  immersed  for  four  to  five  hours,  and  then 
passed  through  70  per  cent,  alcohol  (twenty-four  hours), 
strong  alcohol  (some  days),  absolute  alcohol  (four  to  five 
days).  They  are  then  fit  for  cutting.  The  advantage  of 
the  process  is  said  to  be,  amongst  others,  that  segmentation 
spheres  and  nuclei  are  perfectly  fixed,  the  ova  do  not  become 
porous,  and  cut  like  cartilage. 

For  a  special  formula  for  embryological  purposes,  see 
the  paper  quoted. 

This  liquid  has  been  for  a  long  time  in  great  vogue  not 
only  for  embryological  purposes,  but  for  general  work  and 
cytological  work.  But  opinions  are  divided  as  to  its  merits. 
I  myself  have  extensively  used  it  for  preparing  specimens 
for  dissection  and  for  museum  specimens,  and  have  found  it 
admirable  for  these  purposes.  But  specimens  made  to  test 
its  value  from  a  cytological  point  of  view  have  given  me  only 
second-rate  results. 

MAYER  contributes  the  following  note  on  this  subject  to 
the  Grundzuge  (p.  34)  : — "  Perenyi's  mixture  does  not  appear 
to  have  been  hitherto  considered  from  a  chemical  point  of 
view.  It  is,  however,  easy  to  see  that  as  soon  as  the  mixture 
has  become  violet,  the  chromic  acid  no  longer  exists  in  it  as 
such,  but  has  been  changed  into  chromic  oxide.  At  the 
expense  of  this  oxide  the  alcohol  becomes  oxidised,  and  in 
consequence  of  the  presence  of  the  nitric  acid  becomes 
partially  converted  into  nitric  ether.  Consequently  the 
liquid  is  reduced  essentially  to  a  mixture  of  alcohol  of  at 
most  30  per  cent,  strength  with  about  5  per  cent,  of  nitric 
acid.  An  analogous  mixture  made  by  omitting  the  chromic 
acid  preserves,  according  to  my  experiments,  in  just  the 
same  way  as  Perenyi's,— that  is,  just  as  so  weak  an  acid 
alcohol  can  be  expected  to  preserve,  and  that  is  rather  ill 
than  well.  Objects,  it  is  true,  do  not  shrink  in  it ;  indeed, 
they  rather  swell,  sometimes  to  a  marked  degree.  And,  in 
fact,  observers  are  not  wanting  who  entirely  reject  it  for  the 
fixation  of  ova.  See  the  strange  results  of  Cholodkovsky  in 
the  embryology  of  Blatta,  on  which  I  have  commented  in 
Zool.  Jahresbvricht,  1891  (Arthropoda,  p.  61),  and  which 
Wheeler  and  Heymons  have  later  (ibid.,  1893,  p.  71,  and 


48  CHAPTEE    IV. 

1895,  p.  61)  expressly  referred  to  the  fixing  liquid.  In  any 
case  the  end  is  more  simply  attained  by  taking  simply  acid 
alcohol,  as  recommended  by  me  so  long  ago  as  1880  (Mitth. 
Z.  Stat.  Neapel,  ii,  p.  7),  which  may  be  taken  weaker  or 
stronger  according  to  the  nature  of  the  objects. " 

53.  Chromic  Acid  and  Platinic  Chloride    (MERKEL'S   Macula 
lutea  dey  Menschen,  Leipzig,  1870,  p.  19). — Equal  volumes  of 
1-400  solution  of  chromic  acid  and  1*400  solution  of  platinic 
chloride    (PtClJ.       Objects  should  remain  in  it  for    several 
hours   or   even    days,  as   it   does    not    harden    very   rapidly. 
After  washing  out  with  alcohol  of   50  per  cent,  to   70   per 
cent.,    objects    stain    excellently,    notwithstanding    the    ad- 
mixture of  chromic  acid.      If  objects  that  have  been  fixed 
by  osmic  acid  be  put  into  it  for  some  hours,  blackening  is 
said  to  be  effectually  prevented. 

This  is  an  excellent  hardening  medium  for  delicate 
objects.  Merkel  states  that  he  allowed  from  three  to  four 
days  for  the  action  of  the  fluid  for  the  retina  ;  for  Annelids 
Eisig  employs  an  immersion  of  three  to  five  hours,  and 
transfers  to  70  per  cent,  alcohol  ;  for  small  leeches  Whit- 
man finds  one  hour  sufficient,  and  transfers  to  50  per  cent, 
alcohol. 

Whitman  recommends,  for  the  hardening  of  pelagic  fish 
ova,  a  stronger  mixture  (due,  I  believe  to  Eisig),  viz. — 

0'25  per  cent,  solution  of  platinum  chloride      .       I  vol. 

1  per  cent,  solution  of  chromic  acid          .  1    ,, 

The  ova  to  remain  in  it  one  or  two  days  (WHITMAN,  Methods 
in  Micro.  Anat.,  p.  153). 

Salts. 

54.  Chromates. — The  chromates  are  amongst  the  oldest  and 
best  tried  of  hardening  agents.      The   bichromate  of  potash 
especially  was  at  one  time  universally  employed  for  harden- 
ing all   sorts    of   tissues,   and    a   great  amount  of  classical 
work  has  been  done  with  it. 

About  eighteen  years  ago,  however,  bichromate  fell  into 
disrepute  in  consequence  of  a  criticism  on  its  action  made 
by  FLEMMING.  FLEMMING  pointed  out  (Arch.  f.  mik.  Anat., 
xviii,  J880,  p.  352)  that  though  it  preserves  cytoplasm  well 


FIXING    AND    HARDENING   AGENTS.  49 

it  causes  chromatin  to  swell,  and  therefore  should  not  be 
employed  for  the  study  of  nuclei.  His  readers,  plus  royalistes 
que  le  rui,  took  that  to  be  a  reason  for  abandoning  it 
altogether,  and  from  that  time  until  quite  recently  it  has 
lain  in  the  cold  shadow  of  neglect  forv3lmost  all  purposes 
except  the  hardening  of  nervous  tissuer  Erroneously,  for, 
duly  corrected  with  acetic  acid,  it  affords  a  Correct  xai^  fine 
fixation  of  nuclei ;  whilst  preserving  hyaloplasm  |9$d  its 
inclusions,  secretions,  etc.,  much  better  than  chrd&jgx^Md 
(sometimes  overmuch). 

For  an  elaborate  study  of  the  action  of  chrome  salts  011 
nucleus  and  cytoplasm,  see  BUKCKHAKDT,  La  Cellule,  xii, 
2,  1897,  p.  335.  He  finds  that  the  bichromates  of  sodium, 
ammonium,  magnesium,  strontium,  and  zinc  have  the  same 
destructive  action  on  nuclei  that  the  bichromate  of  potassium 
has ;  but  that  the  bichromates  of  barium,  calcium,  and 
copper  have  not.  The  practical  results  of  his  researches 
may  be  summed  up  as  follows  :  Acetic  acid  ought  always  to 
be  added,  not  only  to  ensure  the  correct  fixation  of  nuclei, 
but  also  to  enhance  penetration  and  the  good  preservation 
of  cytoplasm. 

The  following  is  recommended  by  him  as  a  good  combina- 
tion for  the  fixation  both  of  cytoplasm  and  nucleus  : 

Bichromate  of  barium,  4  per  cent,  solution      .  60  vols. 
Bichromate  of  potassium,  5  per  cent,  solution  .  30     „ 
Glacial  acetic  acid        .  .  .  .  .     5     „ 

(Instead  of  the  barium  you  may  take  4  per  cent,  solution 
of  bichromate  of  calcium,  or  6  per  cent,  solution  of  bichro- 
mate of  copper.) 

For  the  demonstration  of  the  achromatic  figure  of  cell 
division  he  recommends — 

Chromic  acid,  1  per  cent,  solution  .  .  60  vols. 

Bichromate  of  potassium,  5  per  cent,  solution  .  30    „ 
Glacial  acetic  acid        .  .  .  .  .     5     „ 

55.  Bichromate  of  Potash. — Perhaps  the  most  important  of 
all  known  hardening  agents,  sensu  stricto.  It  hardens 
slowly,  much  more  so  than  chromic  acid,  but  it  gives  an  in- 
comparably better  consistency  to  the  tissues,  and  it  has  not 
the  same  tendency  to  make  them  brittle  if  the  reaction  be 

4 


50  CHAPTER  IV. 

prolonged.  They  may  remain  almost  indefinitely  exposed 
to  its  action  without  much  hurt. 

The  strength  of  the  solutions  employed  is  from  2  to  5  per 
cent.  As  with  chromic  acid,  it  is  extremely  important  to 
begin  with  weak  solutions  and  proceed  gradually  to  stronger 
ones.  About  three  weeks  will  be  necessary  for  hardening  a 
sheep's  eye  in  solutions  gradually  raised  from  2  to  4  per  cent. 
Spinal  cord  requires  from  three  to  six  weeks ;  a  brain,  at 
least  as  many  months. 

After  hardening,  the  objects  should  be  well  soaked  out  in 
water  before  being  put  into  alcohol.  They  had  better  be 
kept  in  the  dark  when  in  alcohol  (see  above,  §  41)  (Bonn 
and  OPPEL  [Taschenbuch,  3  Auf.,  1896,  p.  22]  fix  in  the 
dark).  If  you  wish  to  have  a  good  stain  witJi  carmine  you 
should  not  put  the  objects  into  alcohol  at  all,  even  for  a 
second,  until  they  have  been  stained. 

You  may  stain  either  with  carmine  or  hsematoxylm. 

Bichromate  objects  have  an  ugly  yellow  colour  which  cannot  be  removed 
by  soaking  in  water.  It  is  said  that  it  can  be  removed  by  washing  for  a 
few  minutes  in  a  1  per  cent,  solution  of  chloral  hydrate.  Gierke,  however, 
says  that  this  treatment  is  prejudicial  to  the  preservation  of  the  tissues. 

Prof.  GILSON  writes  me  that  alcoholic  solution  of  sulphurous  anhydride 
(S02)  is  very  convenient  for  the  rapid  decoloration  of  bichromate  objects. 
A  few  drops  suffice.  See  also  §§  40  and  41,  and  "  Bleaching." 

To  facilitate  staining  with  hsematoxylin,  WOLFF  (Zeit.f.  wiss.  Mik.,  xv, 
3,  1899,  p.  311)  first  stains  in  Boehmer's  haematoxylin  for  twenty-four 
hours,  and  then  for  a  few  minutes  in  the  same  hsematoxylin  to  which  has 
been  added  1  drop  per  watch-glassful  of  5  per  cent,  solution  of  oxalic  acid. 

The  simple  aqueous  solution  of  bichromate  is  hardly  to  be 
recommended  as  &  fixing  agent,  because  not  only  does  it  not 
preserve  nuclei  (though  it  preserves  cytoplasm)  as  explained 
in  the  last  section,  but  also  because  it  penetrates  very  slowly. 
The  first  of  these  defects  may  be  overcome  entirely,  the 
second  to  some  extent  by  addition  of  acetic  acid  ;  whence 
the  liquid  of  TELLYESNICZKY,  next  §. 

56.  Acetic  Bichromate  (TELLYESNICZKY,  Arch.  f.  mik.  Anat., 
Hi,  2,  1899,  p.  242). — After  a  comparative  study  of  all  the 
usual  fixing  agents,  Telly esniczky  concludes  that  the  two 
best  preservatives  of  cytoplasm  are  osmic  acid  and  bichromate 
of  potash ;  they  are  indeed  the  only  agents  that  fix  in  such  a 
manner  as  to  ensure  the  subsequent  insolubility  of  the  fixed 


FIXING   AND    HARDENING   AGENTS.  51 

struct  tiral  elements.  He  finds  also  that  the  addition  of 
acetic  acid  to  bichromate  not  only  suffices  to  ensure  correct 
fixation  of  nuclei  (see  §  55),  but  also  is  favourable  as  regards 
its  action  on  cytoplasm.  He  recommends  the  following 
formula,  it  being  understood  that  the  proportions  may  be 
varied  if  desired  : 

Bichromate       .  .  .  .  .3  grms. 

Glacial  acetic  acid     ....       5  c.c. 

Water 100     „ 

Smaller  objects  to  remain  in  the  fluid  for  one  or  two  days, 
larger  ones  longer.  Wash  well  in  plenty  of  water,  and  pass 
through  alcohols  of  increasing  strength,  beginning  with  15 
per  cent.  The  results  may  be  compared  with  those  of  liquid 
of  Zenker,  with  the  advantage  that  the  ulterior  treatment  is 
greatly  simplified. 

The  mixtures  of  bichromate  with  osmic  acid  have  been  given  above, 
§§  48  and  49. 

57.  MULLEB'S  Solution. — 

Bichromate  of  potash         ....     2  — 2^  parts. 
Sulphate  of  soda        .....  1    part. 

Water 100    parts. 

The  duration  of  the  reaction  is  about  the  same  as  with  the  simple  solu- 
tion of  chromic  salt. 

This  fluid  was  very  highly  in  vogue  for  many  years,  but  seems  lately  to 
be  much  less  used.  Recent  authors  find  its  action  to  be  identical  with  that 
of  plain  bichromate,  and  doubt  whether  the  sulphate  in  it  has  any  effect 
whatever  as  regards  its  hardening  properties.  I  fancy,  however,  that  the 
superiority  of  this  mixture  over  the  simple  bichromate  solution  is  not 
illusory,  and  is  due  to  the  formation  in  it  of  a  trace  of  free  chromic  acid. 
Fol  says  that  for  mammalian  embryos,  for  which  it  has  been  recommended, 
it  is  worthless. 

58.  ERLICKI'S  Solution  ( Warschauer  med.  Zeit.,  xxii,  Nos.  15 
and  18  (Progres  Medical,  1897,  No.  39).— 

Bichromate  of  potash     .  .  .         2'5  parts. 

Sulphate  of  copper          .  .  .          TO  part. 

Water  .  .  .  .  .      1 00 '0  parts. 

Here  the  addition  of  the  cupric  sulphate  is  intelligible. 
This  salt  is  itself  a  hardening  agent  of  some  energy,  and  may 
well  serve  to  reinforce  the  somewhat  slow  action  of  the 
bichromate.  As  a  matter  of  fact,  "  Erlicki  "  hardens  very 
much  more  rapidly  than  either  simple  bichromate  or  Miiller's 


52  CHAFTBK    IV. 

solution.  A  spinal  cord  may  be  hardened  in  it  in  four  days 
at  the  temperature  of  an  incubator,  and  in  ten  days  at  the 
normal  temperature  (FoL,  Lehrb.  d.  vergl.  mik.  Anat.,  p.  106). 
I  believe  it  to  be  one  of  the  best  hardening  agents  known  for 
voluminous  objects.  Human  embryos  of  several  months  may 
be  conveniently  hardened  in  it. 

Nerve-centres  that  have  been  hardened  in  Erlicki's  fluid  frequently  con- 
tain dark  spots  with  irregular  prolongations,  simulating  ganglion-cells. 
These  were  at  one  time  taken  to  be  pathological  formations,  but  they  are 
now  known  to  consist  of  precipitates  formed  by  the  action  of  the  hardening 
fluid.  They  may  be  removed  by  washing  with  hot  water,  or  with  water 
slightly  acidified  with  hydrochloric  acid,  or  by  treating  the  specimens  with 
0'5  per  cent,  chromic  acid  before  putting  them  into  alcohol  (TscniscH, 
Virchow's  Arch.,  Bd.  xcvii,  p.  173  :  EDINGEB,  Zeit.f.  wiss.  Mik.,  ii,  2,  p.  245  ; 
LOEWENTHAL,  Rev.  med.  de  la  Suisse  romande,  6me  annee,  i,  p.  20). 

59.  Bichromate   and    Oupric   Sulphate  or  Sublimate  Mixture 
(KULTSCHITZKY,    Zeit.  f.   wiss.  Mik.,  iv,  3,  1887,  p.  348). — A  saturated 
solution  of  bichromate   of  potash  and  sulphate  of  copper  in  50  per  cent, 
alcohol,  to  which  is  added  at  the  instant  of  using  a  little  acetic  acid,  five  or 
six  drops  per  100  c.c. 

To  make  the  solution,  add  the  finely  powdered  salts  to  the  alcohol  in 
excess,  and  leave  them  together  in  total  darkness,  for  twenty-four  hours. 

Fix  for  twelve  to  twenty-four  hours  in  the  dark,  otherwise  the  salts  will 
be  precipitated.  Then  treat  with  strong  alcohol  for  twelve  to  twenty-four 
hours,  and  make  sections. 

More  recently  (Arch.f.  mik.  Anat.,  xlix,  1897,  p.  8),  KULTSCHITZKY  re- 
commends a  mixture  of  two  parts  bichromate,  £  part  corrosive  sublimate, 
50  parts  2  per  cent,  acetic  acid,  and  50  parts  96  per  cent,  alcohol.  As 
part  of  the  bichromate  precipitates,  the  mixture  should  be  filtered  after 
twenty-four  hours.  Tissues  of  vertebrates  may  remain  in  it  for  four  to  six 
days. 

60.  Bichromate  of  Ammonia. — A  review  of  the  literature  of  the  sub- 
ject shows  that  this  salt  is  in  considerable  favour  for  hardening,  for  what 
precise  motive  is  not  apparent.     Its  action  is  very  similar  to  that  of  the 
potassium  salt.     Fol  says  that  it  penetrates  somewhat  more  rapidly,  and 
hardens   somewhat    more  slowly.     It  should    be  employed    in   somewhat 
stronger  solutions,  up  to  5  per  cent. 

61.  Neutral  Chromate  of  Ammonia  is  preferred  by  some  anatomists. 
It  is  used  in  the  same  strength  as  the  bichromate.    Klein  has  recommended 
it  for  intestine,  which  it  hardens,  in  5  per  cent,  solution,  in  twenty-four 
hours. 

62.  Bichromates  and  Alcohol. — Mixtures  of   either  bichromate   of 
potash  or  of  ammonia  with  alcohol  may  be  employed,  and  have  a  more  rapid 
action  than  the  aqueous  solution.     Thus  HAMILTON  takes  for  hardening 


FIXING   AND   HARDENING   AGENTS.  53 

brain  a  mixture  of  1  part  methylated  spirits  with  3  parts  of  solution  of 
Miiller  (see  the  chapter  on  the  Central  Nervous  System  in  Part  II ;  see  also 
KULTSCHITZKY'S  Mixture,  ante,  §  59).  Preparations  should  be  kept  in  the 
dark  during  the  process  of  hardening  in  these  mixtures. 

63.  Cupric  Sulphate. — Not  of  general  utility.   See  "  Siphonophora." 

64.  Alum. — Alum  has  been  used  for  fixing  purposes.    After  an  extended 
experience  of  it,  I  only  quote  it  in  order  to  recommend  that  it  be  avoided  at 
all  costs. 

65.  Sulphurous  Acid. — WADDINGTON  (Journ.  Roy.  Mic.  Soc.,  1883, 
p.  185)  uses  a  saturated  solution  of  sulphurous  acid  in  alcohol  for  fixing 
infusoria.     OVEETON  (Zeit.  f.  iviss.  Mik.,  vii,  1890,  p.  9)  uses  an  aqueous 
solution  for  fixing  algae. 


CHAPTER  V. 

FIXING  AND   HARDENING  AGENTS.       CHLORIDES,   ORGANIC 
ACIDS,    AND   OTHERS. 

Chlorides. 

66.  Bichloride  of  Mercury  (Corrosive  Sublimate). — Corrosive 
sublimate  is  stated  in  the  books  to  be  soluble  iu  about  sixteen 
parts  of  cold  and  three  of  boiling  distilled  water.  It  will 
probably  be  found  that  the  aqueous  solution  contains  from  6 
to  7  per  cent,  of  the  sublimate  at  the  temperature  of  the 
laboratory.  It  is  more  soluble  in  alcohol  than  in  water,  and 
still  more  so  in  ether.  Its  solubility  in  all  these  menstrua 
is  augmented  by  the  addition  of  hydrochloric  acid,  ammonious 
chloride,  or  camphor.  With  sodium  chloride  it  forms  a  more 
easily  soluble  double  salt ;  hence  sea-water  may  dissolve  over 
15  per  cent.,  and  hence  the  composition  of  the  liquid  of 
Lang.  v 

The  simple  aqueous  solutions  frequently  deteriorate  in  even  a  short  time 
through  the  formation  of  a  pulverulent  precipitate.  The  nature  of  this 
precipitate  is  unknown  to  me,  and  I  have  been  unable  to  find  any  certain 
means  of  preventing  its  formation.  Thinking  that  it  may  be  due  in  part  to 
ammonia  derived  from  the  air,  I  have  lately  been  in  the  habit  of  adding  a 
little  nitric  acid  to  my  solutions,  and  certainly  have  found  that  they  thus 
keep  much  better.  In  any  case,  for  work  in  which  it  is  desired  to  obtain 
as  energetic  a  fixing  action  as  possible,  it  is  well  to  use  only  freshly  made 
up  solutions.  And  distilled  water  must  always  be  employed  for  making 
up  the  solutions.  The  simple  aqueous  solution  should  give  an  acid  reaction 
with  litmus  paper,  whilst  that  made  with  strong  sodium  chloride  solution 
is  neutral. 

For  fixing,  corrosive  sublimate  may  be,  and  very  frequently 
is,  used  pure  ;  but  in  most  cases  a  finer  fixation  will  be 
obtained  if  it  be  acidified  with  acetic  acid,  say  about  1  per 
cent,  of  the  glacial  acid.  I  find  that  a  saturated  solution  in 
5  per  cent,  glacial  acetic  acid  is  a  very  good  formula.  VAN 


FIXING    AND   HARDENING   AGENTS.  55 

BEN  EDEN  has  recommended  a  saturated  solution  in  25  per 
cent,  acetic  acid,  and  Lo  BIANCO  (Mitth.  Zool.  Stat.  Neapel, 
ix,  1890,  p.  443)  a  mixture  of  2  parts  saturated  solution  with 
1  part  of  49  per  cent,  acetic  acid. 

It  is  sometimes  advisable  to  take  the  most  concentrated 
solution  obtainable.  The  cold  saturated  aqueous  solution 
will  suffice  in  most  cases;  but  for  some  very  contractile 
forms  (coral  polypes,  Planaria),  a  concentrated  solution  in 
warm  or  even  boiling  water  should  be  employed.  For 
Arthropoda  alcoholic  solutions  are  frequently  indicated. 
Delicate  objects,  however,  may  ?/equire  treatment  with  weak 
solutions. 

Objects  should  in  all  cases  be  removed  from  the  fixing  bath 
as  soon  as  fixed,  that  is,  in  other  words,  as  soon  as  they  are 
seen  to  have  become  opaque  throughout,  which  is  practically 
as  soon  as  they  are  penetrated  by  the  liquid.  Small  objects 
are  fixed  in  a  few  minutes.  I  have  found  that  a  "  salivary  " 
gland  of  the  larva  of  Chironomus  is  thoroughly  fixed  in  three 
seconds. 

Wash  out  with  water  or  with  alcohol.  I  consider  alcohol 
almost  always  preferable.  Alcohol  of  about  70  per  cent,  may 
be  taken.  The  extraction  of  the  sublimate  is  hastened  by  the 
addition  of  a  little  camphor  to  the  alcohol.  Or,  much  better 
(MAYER,  Intern.  Afonatxschr.  Anat.  Phys.,  iv,  1887,  p.  43), 
a  little  tincture  of  iodine  may  be  added  to  the  liquid,  either 
alcohol  or  water,  used  for  washing,  enough  to  make  it  of  a 
good  port  wine  colour,  and  the  liquid  be  changed  until  it  no 
longer  becomes  discoloured  by  the  objects.  APATHY  (Jtftfero- 
technik,  p.  148)  takes  a  0'5  per  cent,  solution  of  iodine  in 
strong  alcohol,  leaves  the  objects  in  it  (suspended)  until  they 
have  become  of  about  the  colour  of  the  solution,  and  then 
washes  for  twenty- four  hours  in  pure  alcohol. 

In  obstinate  cases  solution  of  iodine  in  iodide  of  potassium  may  be  taken. 
MATER  (Zeit.  f.  wiss.  Mik.,  xiv,  1897,  p.  28)  makes  it  by  dissolving 
5  grammes  of  iodide  of  potassium  in  5  c.c.  of  distilled  water  £,nd  mixing 
this  with  a  solution  of  O5  gramme  of  iodine  in  45  c.c.  of  90  per  cent, 
alcohol,  but  seldom  uses  the  mixture  concentrated,  merely  adding  as  much 
of  it  as  is  required  to  the  alcohol  or  water  containing  the  objects.  The 
iodine  may  be  washed  out  in  obstinate  cases  with  magnesia  water.  Simi- 
larly APATHY,  Mitth.  Zool.  Stat.  Neapel,  xii,  1897,  pp.  729,  730. 

It  has  been  objected  to  this  process  that  iodine  in  potassic  iodide  precipi- 


56  CHAPTER    V. 

tates  corrosive  sublimate  instead  of  dissolving  it.     That  is  true,  but  the 
precipitate  is  soluble  in  excess  of  the  precipitant. 

It  is  important  that  the  sublimate  be  thoroughly  removed 
from  the  tissues,  otherwise  they  become  brittle,  and  will  not 
stain  so  well.  They  will  also  become  brittle  if  they  are  kept 
long  in  alcohol. 

It  may  happen  that  if  the  extraction  of  the  excess  of  subli- 
mate from  the  tissues  in  bulk  has  been  insufficient,  crystals 
(of  some  mercurial  compound)  may  form  in  the  sections  after 
they  have  been  mounted  in  balsam.  This  may  easily  be  pre- 
vented by  treating  the  sections  themselves  with  tincture  of 
iodine  for  a  quarter  of  an  hour  before  mounting.  This  will 
do  away  with  the  necessity  of  treating  the  tissues  in  bulk 
with  iodine,  which  is  frequently  a  very  long  process  (unless 
it  is  desired  to  keep  the  material  for  a  long  time  in  alcohol 
before  making  the  sections). 

MANN  (Zeit.  f.  wiss.  Mik.,  xi,  1894,  p.  479)  prefers  treat- 
ing the  sections  rather  than  the  tissues  in  bulk,  on  the 
ground  that  the  iodine  makes  them  soft,  so  that  they  shrink 
on  coming  into  paraffin.  SCHAPEE  (Anat.  Anz.,  xiii,  1897, 
p.  463),  on  the  other  hand,  has  shown  that  neglect  to  extract 
the  sublimate  from  the  tissues  in  bulk  may  give  birth  to 
serious  artefacts,  which  appear  to  arise  during  the  imbedding 
process. 

You  may  stain  in  any  way  you  like.  Carmine  stains  are 
peculiarly  brilliant  after  sublimate. 

It  must  be  remembered  that  the  solutions  must  not-  be 
touched  with  iron  or  steel,  as  these  produce  precipitates  that 
may  hurt  the  preparations.  To  manipulate  the  objects,  wood, 
glass  or  platinum  may  be  used  ;  for  dissecting  them,  hedge- 
hog spines,  or  quill  pens,  or  cactus  spines. 

When  properly  employed,  sublimate  is  for  general  work 
undoubtedly  a  most  useful  fixing  agent.  It  is  applicable  to 
most  classes  of  objects.  It  is  perhaps  less  applicable,  in  the 
pure  form,  to  Arthropods,  as  it  possesses  no  great  power  of 
penetrating  chitin.  For  cytQlogical  work  it  is,  according  to 
my  experience,  not  to  be  trusted,  and  only  to  be  recommended 
where  more  precise  fixing  agents,  such  as  solution  of  Flem- 
ming,  are  counter-indicated  by  reason  of  their  lack  of  pene- 
tration, or  the  like.  Amongst  other  defects  it  has  that  of 
frequently  causing  serious  shrinkage  of  cells. 


FIXING    AND    HARDENING    AGENTS.  57 

67.  Corrosive  Sublimate  (LANG'S  formula,  ZooL  Anzeiger,  1878,  i, 
p.  14).     For  Planaria.— Take- 
Distilled  water      .         .         .     100  parts  by  weight. 
Chloride  of  sodium        .         .         6  to  10  parts. 

Acetic  acid  .  .  .  .  6  to  8  „ 
Bichloride  of  mercury  .  .  3  to  12  ,, 
(Alum,  in  some  cases  .  .  T».) 

Second  formula  (ibid.,  1879,  ii,  p.  46). — Make  a  concentrated  solution  of 
corrosive  sublimate  in  picro-sulpiiuric  acid,  to  which  has  been  added  5  per 
rent,  of  acetic  acid. 

68.  Other  Simple  Aqueous  Solutions. — A  solution  containing  5  g. 
sublimate,  O'o  g.  sodium  chloride,  and  100  c.c.  water,  has  been  quoted  as 
"  solution  of  GAULE." 

KAISEE'S  solution  consists  of  10  g.  sublimate,  3  g.  glacial  acetic  acid, 
and  300  g.  distilled  water  (from  Zeit.f.  wiss.  Mik.,  xi,  3,  p.  378). 

M.  HEIDEXHAIX  has  recommended  a  0'5  per  cent,  solution  of  sodium 
chloride  saturated  while  hot  with  sublimate. 

69.  Alcoholic   Solutions. — APATHY    (Mikrotechnikj   p.    Ill) 
writes  that  he  thinks  that  "  a  solution  of  3  to  4  grammes  of 
sublimate  and  0*5  gramme   sodium  chloride  in  50  per  cent, 
alcohol  "  (quantity  not  stated  !)  will  prove  to  be  "  for  most 
objects  the  best  of  fixatives  for  general  purposes." 

For  CARNOY'S  and  OHLMACHER'S  alcoholic  fluids,  see  §  84. 

69a.  HELD  (Arch.  f.  Anat.  u.  Phys.,  Abth.  1897,  p.  227, 
fixes  nerve  tissue  in  a  1  per  cent,  solution  of  sublimate  in  4 
per  cent.  Aceton,  and  washes  out  through  increasingly-con- 
centrated grades  of  aceton. 

70.  Mercuro-nitric  Mixtures. — FICKNZEL  (Arch.f.  mik.  Anat., 
xxvi,    1885,  p.    232)   recommends   a  half-saturated    solution 
of  sublimate  in  80  per  cent,  alcohol,  to  which  is  added  nitric 
acid  in  the  proportion  of   1  drop  to  1  c.c.  or  2  c.c.      Objects 
of  the  size  of  a  pea  to  be  fixed  in  it  for  five  or  ten  minutes, 
then  hardened  (?  how  long)   in  the   same  sublimate  alcohol 
without  the  acid,  and  finally  in  90  per  cent,  alcohol.      It  is 
said  that  the  nitric  acid  renders  after-treatment  with  iodine 
unnecessary. 

GILSON'S  Mixture. — I  am  indebted  to  Prof.  GILSON  for 
kindly  sending  his  latest  formula  (1895),  which,  is  as  follows 
(I  have  simplified  it  by  omitting  one  place  of  decimals)  : 


58  CEAPTEE   V. 

Nitric  acid  of  46°  strength  (this 
would  be  sp.  gr.  1*456,  or  80  per 
cent.,  nearly)  .  .  .  .15  c.c. 

Glacial  acetic  acid          .  .  .        4    ,, 

Corrosive  sublimate       .  .  .20  grins. 

60  per  cent,  alcohol       .  .  .    100  c.c. 

Distilled  water     ....    880    „ 

When  required  for  marine  animals  add  a  few  crystals  of 
iodine,  which  will  prevent  the  formation  of  precipitates  of 
sea  salts.  If  in  any  case  the  preparations  should  show  a 
granular  precipitate,  due  probably  to  an  abundance  of 
phosphates  in  the  tissues,  the  precipitate  may  be  removed 
by  washing  with  water  containing  a  little  tincture  of  iodine. 

I  have  tried  this  mixture  and  find  that  it  affords  in 
general  a  faithful  and  delicate  fixation,  and  gives  to  tissues 
an  excellent  consistency.  Objects  may  remain  in  it  for  a 
considerable  time  without  hurt.  Tissues  are  left  in  a  state 
very  favourable  for  staining.  The  liquid  has  a  high  degree 
of  penetration.  A  treatment  for  a  few  days  with  it  will 
serve  to  remove  the  albumen  from  the  ova  of  Batrachians. 
This  liquid  may  be  recommended  to  beginners,  as  it  is 
very  easy  to  work  with.  For  some  objects,  as  I  found, 
the  proportion  of  sublimate  may  be  increased  with  ad- 
vantage. 

CARAZZI  (Mitth.  Zool.  Stat.  Neapel,  xii,  1896,  p.  381) 
takes  one  litre  of  1  per  cent,  sodium  chloride  solution  and 
adds  to  it  20  grammes  of  sublimate,  dissolved  in  100  c.c.  of 
70  per  cent,  alcohol,  15  c.c.  of  concentrated  nitric  acid,  and 
5  c.c.  'of  glacial  acetic  acid.  Very  small  objects  to  be  fixed 
for  one  to  two  hours,  larger  ones  four  to  six.  Wash  out  in 
iodine-alcohol  for  two  to  twenty-four  hours. 

KOSTANECKI  and  SIKDLKCKI  (Arch.  f.  mile.  Anat.,  xlviii, 
1896,  p.  181)  take  a  mixture  of  saturated  sublimate  solution 
and  3  per  cent,  nitric  acid  in  equal  parts,  or  a  mixture  of 
equal  parts  of  sublimate  solution,  3  per  cent,  nitric  acid,  and 
absolute  alcohol,  fix  for  twenty-four  hours,  and  wash  out  in 
iodine-alcohol. 

71.  Picro-sublimate  Mixtures. — RABI/S  (Zeit  f.  wins.  Mik., 
xi,  2,  1894,  p.  165).  Sublimate,  saturated  solution  in  water, 
1  vol.  ;  a  similar  solution  of  picric  acid,  1  vol. ;  distilled 


FIXING    AND    HARDENING  AGENTS.     '  59 

water,  2  vols.  Embryos  may  be  left  in  it  for  twelve  hours, 
washed  for  two  hours  in  water,  and  brought  into  weak 
alcohol. 

MANN'S  (op.  cit.,  xi,  4,  1895,  p.  480). — 1  per  cent,  of  picric  acid  with  or 
without  1  per  cent,  of  tannin  in  a  saturated  solution  of  sublimate  in  normal 
salt  solution. 

The  same  author's  Alcoholic  Picro-sublimate  (Anat.  Am.,  8,  1893, 
pp.  441 — 443)  consists  of  absolute  alcohol  100  c.c.,  picric  acid  4  grms., 
sublimate  15  grms.,  tannin  6  to  8  grms.  The  tannin  is  added  in  order  to 
prevent  excessive  hardening. 

TELLYESNICZKY  (Arch.  f.  mik.  Anat.,  lii,  1898,  p  237)  says  of  Mann's 
tannin  liquid,  "  its  action  is  an  entirely  destructive  one." 

O.  vom  RATB'S  Picro-sublimate  (Anat.  Anz.,  xi,  9,  1895, 
p.  286). — Cold  saturated  solution  of  picric  acid,  1  part;  hot 
saturated  solution  of  sublimate,  1  part ;  glacial  acetic  acid,  | 
to  1  per  cent.  Fix  for  several  hours  and  bring  direct  into 
alcohol . 

The  same  author's  Picro-sublhnate-osmic  Mixture  (loc.  cit.) 
consists  of  the  above  with  the  addition  of  10  per  cent,  of  2 
per  cent,  osmic  acid  solution. 

72.  Osmio-sublimate  Mixtures. — MANX'S  (Zeit.  f.  wiss.  Milt., 
xi,  4,   1894,  p.  481)  consists  of  a  freshly-prepared  mixture 
of  equal  parts  of  1  per  cent,  osmic  acid  solution  and  saturated 
solution    of    sublimate   in   normal    salt    solution    (for  nerve- 
centres)  . 

For  APATHY'S  see  §  358. 

DRUNER'S  (Jena.  Zeit.  Naturw.,  xxviii,  1894,  p.  294)  con- 
sists of  1  part  of  1  per  cent,  osmic  acid  solution  added  to  20 
parts  of  a  solution  of  5  per  cent,  each  of  sublimate  and 
glacial  acetic  acid  in  water. 

O.  vom  RATH'S,  see  last  §. 

73.  Chromo-sublimate. — Lo     BIANCO     (Mitth.    Zool.     Stat. 
Neapel,  ix,  3,  1890,  p.  443).        Concentrated  sublimate  solu- 
tion, 100  parts,  1  per  cent,  chromic  acid,  50  parts. 

74.  ZEXKER'S  Mixture  (Milnchener  med.  Wochenschr.,  24, 1894,  p.  534 ; 
quoted  from  MERCIER,  Zeit.  f.  wiss.  Mik.,  xi,  4,  1894,  p.  471,  where  will  be 
found  minute  instructions  for  using  it).  Five  per  cent,  of  sublimate  and 
o  per  cent,  of  glacial  acetic  atid,  dissolved  in  solution  of  MULLER.  Fix  for 


60  CHAPTER    V. 

several  hours',  wash  out  with  water,  treat  the  tissues  in  bulk,  or  the  sections 
with  alcohol  containing  tincture  of  iodine. 

TELLYESNICZKY  (Arch.f.  mik.  Anat.,  Hi,  2,  1898,  p.  238),  who  has  lately 
tried  this  fluid  on  testes  of  Salamandra,  writes  that  for  accurate  preserva- 
tion of  cell-structures  it  is  comparable  to  liquid  of  FLEMMING,  with  the 
advantage  of  better  penetration  (therefore  applicability  to  larger  pieces  of 
material),  absence  of  over-fixation  of  superficial  layers,  easier  staining,  and 
cheapness.  He  thinks  the  sodium  sulphate  may  be  omitted  from  it  with- 
out  any  change  in  the  results.  Numerous  recent  authors  speak  very  highly 
of  this  reagent. 


75.  Foi's  Mixture  (Quart.  Journ.  Mic.  Sci.,  1895,  p.  287  ;  Journ.  Roy. 
Mic.  Soc.,  1895,  p.  486). — Equal  parts  of  saturated  solution  of  sublimate 
in  normal  salt  solution,  and  of  liquid  of  Miiller,  or  5  per  cent,  solution  of 
bichromate. 

Very  much  like  Zenker's  mixture,  with  the  acetic  acid  omitted,  which 
appears  to  me  to  be  certainly  for  most  purposes  a  false  step. 

76.  Chromo-nitric    Sublimate   (ROSEXSTADT,  Arch.  f.   mik.   Anat., 
xlvii,  1896,  p.  748). — Three  volumes  of  saturated  aqueous  sublimate  solution 
with  1  volume  of  liquid  of  PEEENYI. 


77.  Chloride  of  Platinum  (Platinic  Chloride,  PtCl4).— A 
reagent,  originally  introduced  for  the  study  of  karyokinesis, 
but  of  general  application.  RABL,  to  whom  we  owe  the 
introduction  of  this  agent,  employed  an  aqueous  solution  of 
1*300.  The  objects  remained  in  it  for  twenty-four  hours, 
and  were  then  washed  with  water,  hardened  in  alcohol,  and 
sectioned. 

Rabl  found  it  give  better  results  (for  the  study  of  karyo- 
kinesis)  than  any  other  reagent  except  chromoformic  acid 
(§  44).  It  causes  a  slight  shrinkage  of  the  chromatin  ele- 
ments, a  condition  that  renders  the  granules  of  Pfitzner  and 
the  longitudinal  division  of  the  elements  very  distinctly 
visible  (see  Rabl's  paper  in  Morph.  Jahrb.,  Bd.  x,  1884,  p. 
216). 

Platinum  chloride  is  an  extremely  deliquescent  salt,  and 
for  this  reason  had  better  be  procured  in  solution.  Ten  per 
cent,  solutions  are  found  in  commerce. 

For  the  platinic  mixtures  see  ante  §  49,  50,  53,  also  RABI/S 
mixture,  under  Embryolor/ical  Method.?.  For  the  platinic 
formol  of  BOUIN.  see  Arch.  d'Anat.  Microsc.,  ii,  4,  1899,  p. 
423. 


FIXING  AND    HARDENING    AGENTS.  61 

78.  Palladium  Chloride  (F.  E.  SCHULTZE,  Arch.  mik.  Anat.,  iii,  1867, 
1>.   477). — This   reagent   was    recommended    by   Schultze  as  a    hardening 
agent,  partly  as  giving  to  tissues  a  better  consistency  than  chromic  acid  or 
Midler's  solution,  and  partly  on  account  of  a  special  faculty  for  penetrating 
organs  rich  in  connective  tissue  that  he  attributed  to  it.     It  is  an  impreg- 
nation  reagent,  staining  certain  elements  of  tissues  in  various  tones  of 
brown.     For  the  somewhat  lengthy  details  of  the  manner  of  employing  it, 
the  reader  is  referred  to  the  paper  quoted. 

CATTANEO  recommends  it,  used  in  solutions  of  1'300,  1*600,  or  1'SOO 
strength,  for  from  one  to  two  minutes,  as  being  the  best  of  fixatives  for 
Infusoria. 

This  salt  is  found  in  commerce  in  the  solid  state.  To  dissolve  it,  take  10 
grammes  of  the  salt,  one  litre  of  water,  and  four  to  six  drops  of  hydrochloric 
acid.  Solution  will  be  effected  in  twenty-four  hours. 

FEENKEL  (Anat.  Anz.,  viii,  1893,  p.  538;  Zeit.f.  wiss.  Mik.,  x,  2,  1893, 
p.  243)  recommends  for  connective  tissue  a  mixture  of  15  parts  1  per  cent, 
palladium  chloride,  5  parts  2  per  cent  osmic  acid,  and  a  few  drops  of  acetic 
acid. 

79.  Iridium  Chloride  (EiSEN,  Zeit.f.  wiss.  Mik.,  xiv,2,  1897,  p.  195). 
— Solution  of  one  half  or  one  fifth  per  cent.,  acidified  with  1  per  cent,  of 
glacial  acetic  acid. 

My  specimens  show  about  the  worst  fixation  I  have  ever  seen.  It  simply 
does  not  fix  at  all. 

80.  Perchloride  of  Iron  (FoL,  Zeit.  f.  wiss.  Zool.,  Bd.  xxxviii,  1883, 
p  491 ;  and  Lehrb.  d.  vergl.  mik.  Anat.,  p.  102). — Fol  recommends  1  vol. 
of  Tinct.  Ferri  Perchlor.  P.  B.  diluted  with  5  to  10  vols.  of  70  per  cent, 
alcohol. 

The  tincture  diluted  with  3  to  4  vols.  of  either  alcohol  or  water  has  been 
recommended  for  fixing  medullated  nerve  by  PLATNER  (Zeit.f.  wiss.  Mik., 
vi,  2,  1889,  p.  187). 

81.  Chloride  of  Zinc   is   sometimes    used   for   hardening    brain   (see 
Part  II).     GILSON  (La  Cellule,  vi,  1,  1890,  p.  122)  has  used  it  with  good 
results  as  a  fixative  for  the  silk-glands  of  Lepidoptera,  as  follows  : 

Glacial  acetic  acid      .....         5  c.c. 
Nitric  acid  of  46°  (or  80  per  cent,  nearly)          5    „ 

Alcohol  of  80  per  cent 100   „ 

Distilled  water 300   „ 

Dry  chloride  of  zinc  .  20  grammes. 

81a.  Fluorides  (MABPMANN),  see  Zeit.  f.  angew.  Mik.,  v,  1899,  p.  33, 
or  Journ.  Roy.  Mic.  6'oc.,  1899,  p.  456. 


62  CHAPTER  V. 


Organic  Acids,  and  other  Agents. 

82.  Acetic  Acid. — The  place  of  honour  amongst  organic 
acids  considered  as  fixing  agents  appears  rightfully  to  belong 
to  this  old-fashioned  reagent.  Flemming,  who  has  made 
a  special  investigation  of  its  action  on  nuclei,  finds  (Zellsub- 
stanz,  etc.,  p.  380)  that  the  best  strength  is  from  0*2  to  1 
per  cent.  Strengths  of  5  per  cent,  and  more  bring  out  the 
nuclein  structures  clearly  at  first,  but  after  a  time  cause 
them  to  swell  and  become  pale,  which  is  not  the  case  with 
the  weaker  strengths  (ibid.,  p.  103).  It  must  now  be 
stated  that,  thanks  to  v.  BENEDEN,  the  strong  acid  has  become 
established  as  a  valuable  fixative  of  certain  objects.  It  is 
particularly  applicable  to  very  contractile  objects,  such  as 
many  Vermes,  Coelenterata,  and  Nudibranchs ;  it  kills  them 
with  the  utmost  rapidity,  and  has  a  tendency  to  leave  them 
fixed  in  the  state  of  extension.  The  modus  operandi  is  in 
general  as  follows : — Pour  glacial  acetic  acid  in  liberal 
quantity  over  the  organisms,  leave  them  until  they  are  pene- 
trated by  it — which  should  be  in  five  or  six  minutes,  as  the 
strong  acid  is  a  highly  penetrating  reagent — and  wash  out 
in  frequent  changes  of  alcohol  of  gradually  increasing 
strength.  Some  persons  begin  with  30  per  cent,  alcohol, 
but  this  appears  to  me  rather  weak,  and  I  think  70  per  cent, 
or  at  least  50  per  cent,  alcohol  should  be  preferred. 

I  see  no  reason  why  other  energetic  reagents  should  not 
be  combined  with  the  glacial  acetic  acid  if  desired.  Dr. 
LINDSAY  JOHNSON  (in.  litt.)  has  found  that  one  of  the  best 
fixatives  for  retina  is  a  mixture  of  equal  parts  glacial  acetic 
acid  and  2  per  cent,  osmic  acid.  S.  Lo  BIANCO  adds  to  the 
"  concentrated  "  *  acid  one  tenth  of  a  1  per  cent,  solution  of 
chromic  acid.  He  finds  that  even  this  small  proportion  of 
chromic  acid  serves  to  counteract  in  a  marked  degree  the 
softening  action  of  the  acetic  acid. 

It  goes  without  saying  that  in  v.  Beneden's  process  the 
acetic  acid  does  not  play  the  part  of  a  fixing  agent  sensu 
stricto,  that  is,  an  agent  that  hardens  cells  at  the  same  time 

*  MAYER,  in  the  Ghrundzugc,  explains  that  the  acid  referred  to  as 
"  concentrated  "  by  Lo  BIANCO  in  his  Metodi  (Mitth.  Zool.  8  at.  Neapel, 
ix,  3,  p.  435;  is  an  acid  of  approximately  49  per  cent  (sp.  gr.  1'060). 


FIXING    AND    HARDENING   AGENTS.  63 

that  it  kills  them.  The  rationale  of  the  process  is  that  the 
acid  kills  the  tissues,  whilst  the  alcohol  comes  in  and  hardens 
them  sufficiently  before  they  have  had  time  to  become  de- 
formed by  the  action  of  the  acid.  Acetic  acid,  used  alone, 
is  only  a  fixative  for  a  limited  time.  If  its  action  be  pro- 
longed and  not  controlled  by  the  action  of  some  other  agent, 
it  becomes  a  swelling  agent.  Its  function  in  mixtures  is, 
besides  that  of  killing,  the  valuable  one  of  counteracting  the 
shrinking  action  of  the  ingredients  with  which  it  is  com- 
bined, and  by  its  swelling  action  enhancing  the  penetration 
of  the  mixture ;  whilst  by  clarifying  tissues  it  aids  in  the 
optical  differentiation  of  their  elements.  For  these  reasons 
it  is,  in  all  cases  in  which  its  presence  is  not  absolutely 
counter-indicated  (connective  tissue,  delicate  calcareous  struc- 
tures, etc.),  a  most  valuable  ingredient,  almost  a  sine  qua  non, 
in  fixing  mixtures. 

The  proportions  in  which  it  should  enter  into  mixtures  in 
general  seem  to  me  to  be  from  0'5  per  cent,  to  5  per  cent,  of 
the  glacial  acid ;  higher  strengths,  such  as  25  per  cent,  to 
100  per  cent.,  being  only  indicated  in  cases  in  which  the 
highest  possible  penetration  is  the  chief  consideration. 

Throughout  this  work,  wherever  acetic  acid  is  mentioned, 
it  is  the  glacial  acid  that  is  meant  unless  the  contrary  is 
stated. 

All  liquids  containing  a  large  proportion  of  this  acid  (e.  g. 
§§  83,  84)  should  only  be  allowed  to  act  for  a  very  short 
time. 

83.  Acetic  Alcohol  (CARNOY,  La  Cellule,  t.  iii,  1,  1886,  p.  6 ; 
and  ibid.,  1887,  2,  p.  276;  v.  BENEDEN  et  NEYT,  Bull.  Ac. 
roy.  d.  sci.  de  Belg.,  t.  xiv,  1887,  p.  218 ;  ZACHARIAS,  Anat. 
Anz.,  iii,  Jahrg.  1,  1888,  pp.  24 — 27;  v.  GEHUCHTEN,  ibid., 
8,  p.  227). — CARNOY  -has  given  two  formulae  for  this  important 
reagent.  The  first  is — 

Glacial  acetic  acid       .  .  .1  part. 

Absolute  alcohol          .          .          .3  parts. 

The  second  is — 

Glacial  acetic  acid  .  .  .1  part. 

Absolute  alcohol  .  .  .6  parts. 

Chloroform  3 


64  CHAPTER  V. 

The  addition  of  chloroform  is  said  to  render  the  action  of 
the  mixture  more  rapid, 

V.  BENEDKN  and  NKYT  take  equal  volumes  of  glacial  acid 
and  absolute  alcohol. 

ZACHAKIAS  takes  — 

Glacial  acetic  acid        .  .  .1  part. 

Absolute  alcohol  .  .  .4  parts. 

Osmic  acid          .  .  .  .a  few  drops. 

Acetic  alcohol  is  one  of  the  most  penetrating  and  quickly 
acting  fixatives  known.  It  preserves  both  nuclei  and  cyto- 
plasm, and  admits  of  admirable  staining  in  any  way  that  may 
be  preferred.  It  was  employed  by  all  of  the  authors  quoted 
for  the  study  of  karyokinesis  in  the  ova  of  Ascaris  —  pro- 
verbially one  of  the  most  difficult  objects  to  fix,  —  but  from 
what  I  have  seen  of  it  I  should  say  that  it  is  applicable  with 
advantage  to  many  other  objects,  and  has  been  found  to  give 
excellent  results  with  central  nervous  tissue.  You  may  wash 
out  with  alcohol  and  treat  afterwards  in  any  way  that  may  be 
preferred  (aqueous  liquids  being  avoided  as  far  as  possible)  . 
But  the  sublimate  liquid,  next  §,  will  probably  be  found  in 
many  cases  superior. 


84.  Acetic  Alcohol  with  Sublimate.  —  The  following  mixture 
due  to  G-ILSON,  was  first  published  by  CARNOY  and  LHJBRDN 
(La  Cellule,  xiii,   1,   1837,  p.    68),  and   most   highly   recom- 
mended by  them  as  superior  for  ova    of    Ascaris   with  the 
shell  formed  to  the  chloroform  liquid   of  the   last  §.       The 
addition   of   the    sublimate   serves   to    restrain   the   swelling 
action  of  the  acetic  acid,  which  is  insufficiently  done   by  the 
other  ingredients. 

Absolute  alcohol  .  .  .  .1  vol. 

Glacial  acetic  acid          .  .  .  1     „ 

Chloroform  .  .  .  .  .  .      1     „ 

Sublimate  to  saturation. 

Isolated  ova  of  Ascaris,  even  though  furnished  with  a 
shell,  are  fixed  in  twenty-five  to  thirty  seconds.  Entire 
oviducts  take  about  ten  minutes.  The  liquid  is  therefore 
one  of  the  most  penetrating  and  rapidly  acting  of  any,  if  not 
the  most. 

Wash  out  with  alcohol  until  all  traces  of  odour  of  the 
acetic  acid  have  disappeared  (I  myself  wash  out  with  alcohol 


FIXING    AND    HARDENING    AGENTS.  65 

containing  tincture  of  iodine).      I  consider  this  a  very  fine 
reagent. 

OHLMACHEE  (Journ.  Exper.  Medicine,  ii,  6,  1897,  p.  671)  has  arrived 
independently  at  a  sublimate  modification  of  Carney's  original  chloroform 
liquid.  He  takes — 

Absolute  alcohol 80  parts. 

Chloroform 15      „ 

Glacial  acetic  acid        .         .         .  5      ,, 

Sublimate  to  saturation  (about  20  per  cent.). 

"Ordinary  pieces"  of  tissue  are  sufficiently  fixed  in  fifteen  to  thirty 
minutes.  Entire  human  cerebral  hemispheres,  subdivided  by  Meynert's 
section,  take  eighteen  to  twenty-four  hours.  Wash  out  in  iodine  alcohol 
(§  66).  Although  this  liquid  is  historically  a  modified  acetic-alcohol,  yet 
in  view  of  the  relatively  small  amount  of  acid  in  it,  it  should  rather  be 
classed  as  an  alcoholic  sublimate  solution,  §  69. 

85.  Formic  Acid  may  be  used  dilute  in  the  same  way  as  acetic  acid 
(supra,  §  82).  It  is  possible  that  it  might  also  take  the  place  of  acetic 
acid  in  the  concentrated  form,  but  I  am  not  aware  of  any  experiments  in 
this  direction. 

86.  Chloride  and  Acetate  of  Copper  (Rip art  et  Petit' s 
Liquid,  CAKNOY,  La  Biologie  Cellulaire,  p.  94). — 

Camphor  water  (not  saturated)         .   75  grammes. 
Distilled  water       .  .  .  .  75         „ 

Crystallised  acetic  acid  .  .  .1  gramme. 

Acetate  of  copper  .          .          .     0'30  „ 

Chloride  of  copper          .  .  .     0*39  „ 

This  is  a  very  moderate  and  delicate  fixative.  I  consider 
that  it  has  not  sufficient  hardening  power  for  objects  that 
are  intended  to  be  dehydrated  and  mounted  in  balsam,  but 
is  frequently  excellent  and  sometimes  indispensable  for 
objects  that  are  to  be  studied  in  as  fresh  a  state  as  possible 
in  aqueous  media.  Objects  fixed  in  it  stain  instantaneously 
and  perfectly  with  methyl  green.  Osmic  acid  may  be  added 
to  the  liquid  to  increase  the  fixing  action.  For  cytological 
researches  this  is  a  most  invaluable  medium. 

87.  Acetate  of  Uranium  (SCHKNK,  Mitth.  a.  d.  Embryol.  Inst.  Wien, 
1882,  p.  95  ;  cf.  GILSON,  La  Cellule,  i,  1885,  p.  141).— This  reagent  is  very 
similar  in  its  properties  to  picric  acid.  It  has  a  mild  fixing  action,  and  a 
high  degree  of  penetration,  which  may  make  it  useful  for  Arthropoda.  It 
may  be  combined  with  methyl  green,  which  it  does  not  precipitate. 

For  Acetate  of  Lead  see  "  Neurological  Methods,"  Hardening. 

5 


66  CHAPTER    V. 

88.  Iodine. — Iodine  possesses  considerable  hardening  properties,  and  a 
very  high  degree  of  penetration.  KENT  (Manual  of  the  Infusoria,  1881, 
p.  114  ;  Journ.  Boy.  Mic.  Soc.  (N.  S.),  iii,  1883,  p.  730)  has  found  it  to  act 
in  a  manner  almost  identical  with  osmic  acid,  and  in  some  instances  even 
more  efficiently  (for  fixing  Infusoria).  His  instructions  are  as  follows : — 
*'  Prepare  a  saturated  solution  of  potassic  iodide  in  distilled  water,  saturate 
this  solution  with  iodine,  filter,  and  dilute  to  a  brown-sherry  colour.  A  very 
small  portion  only  of  the  fluid  is  to  be  added  to  that  containing  the 
Infusoria." 

Or  you  may  use  LUGOL'S  solution,  of  which  the  formula  is  as  follows  : 

Water 100  parts 

Iodide  of  potassium 6      ,, 

Iodine 4     ,, 

Iodine  certainly  kills  cells  very  rapidly,  without  deforming  them.  Per- 
sonally I  have  found  it  very  useful  for  the  examination  of  spermatozoa. 
Unfortunately  I  am  not  acquainted  with  any  nuclear  stain  that  will  work 
well  with  it. 

Very  small  objects  may  be  instantaneously  fixed  by  means  of  vapour  of 
iodine.  Crystals  of  iodine  may  be  heated  in  a  test-tube  till  the  vapours 
are  given  off  ;  then  on  inclining  the  tube  the  heavy  vapours  may  be  made  to 
flow  over  the  objects  arranged  on  a  slide.  The  slide  should  then  be  warmed 
to  about  40°  C.  for  two  or  three  minutes  in  order  to  evaporate  the  iodine 
'  from  the  objects,  which  may  then  be  mounted  or  otherwise  treated  as  desired 
{OvEBTON,  Zeit.f.  wiss.  Mik.,  vii,  1,  1890,  p.  14). 

Iodine  may  be  used  in  combination  with  alcohol  for  hardening,  and 
render  service  through  its  great  penetrating  power.  See  the  method  of 
BETZ,  post,  Part  II. 

89,  Picric  Acid, — Picric  acid  in  pure  aqueous  solution  should 
always  be  employed  in  the  form  of  a  strong  solution.  (That  is 
to  say,  strong  solutions  must  always  be  employed  when  it  is 
desired  to  make  sections  or  other  preparations  of  tissues 
with  the  elements  in  situ,  as  weak  solutions  macerate  ;  but 
for  dissociation  preparations  or  the  fixation  of  isolated  cells, 
weak  solutions  may  be  taken.  Flemming  finds  that  the 
fixation  of  nuclear  figures  is  equally  good  with  strong  or 
weak  solutions.)  The  saturated  solution  is  the  one  most 
employed.  (One  part  of  picric  acid  dissolves  in  about  86 
parts  of  water  at  15°  C.  •*  in  hot  water  it  is  very  much  more 
soluble.)  Objects  should  remain  in  it  for  from  a  few 
seconds  to  twenty-four  hours,  according  to  their  size.  For 
Infusoria  one  to  at  most  two  minutes  will  suffice,  whilst 
objects  of  a  thickness  of  several  millimetres  require  from 
three  to  six  hours'  immersion. 

*  Benedikt  and  Knecht,  Chemistry  of  the  Coal-tar  Colours,  p.  214. 


FIXING    AND   HARDENING    AGENTS.  67 

Picric  acid  should  always  be  washed  out  with  alcohol,  as 
water  is  hurtful  to  tissues  that  have  been  prepared  in  it. 
For  the  same  reason  during  all  remaining  stages  of  treat- 
ment, water  should  be  avoided  ;  staining  should  be  performed 
by  means  of  alcoholic  solutions,  the  only  exceptions  to  this 
rule  being  in  favour  of  methyl  green,  and  some  few  other 
aqueous  stains  that  are  themselves  weak  hardening  agents, 
such  as  haemalum,  carmalum,  etc. 

It  has  been  found  by  JELINEK  (Zeit.  f.  wiss.  Mik.,  xi,  2, 
1894,  p.  242)  that  the  extraction  of  picric  acid  is  greatly 
quickened  by  the  addition  of  a  base  to  the  wash-alcohol. 
He  recommends  carbonate  of  lithia.  A  few  drops  of  a 
saturated  solution  of  the  salt  in  water  are  added  to  the 
alcohol ;  a  slight  precipitate  is  formed.  The  objects  are  put 
into  the  turbid  alcohol,  which  becomes  clear  and  yellow  in 
proportion  as  the  picrin  is  extracted.  Furthur  quantities  of 
carbonate  are  added  from  time  to  time  until  the  colour  has 
been  entirely  extracted  from  the  tissues. 

Tissues  fixed  in  picric  acid  can,  after  removal  of  the  acid 
by  soaking,  be  perfectly  stained  in  any  stain.  It  is  not 
generally  necessary  to  remove  the  picric  acid  by  washing 
out  before  staining.  Mayer's  paracarmine,  Grenadier's  alco- 
holic borax-carmine,  or  Mayer's  haemacalcium  may  be  recom- 
mended for  entire  objects. 

The  most  important  property  of  picric  acid  is  its  great 
penetration.  This  renders  it  peculiarly  suitable  for  the  pre- 
paration of  chitinous  structures.  For  such  objects  alcohol 
of  70  per  cent,  to  90  per  cent,  should  be  taker,  for  washing 
out,  and  staining  should  be  done  by  means  of  Mayer's 
cochineal  or  haemacalcium. 

89a.  Picro-acetic  Acid. — Saturated  solution  of  picric  acid  in 
1  per  cent,  acetic  acid  has  given  me  better  preparations  than 
those  obtained  by  any  other  picric  liquid,  except  picro-aceto- 
osmic. 

BOVEEI  (Zellenstndien,  1,  1887,  p.  11)  dilutes  a  concentrated  aqueous 
solution  with  two  volumes  of  water  and  adds  1  per  cent,  of  acetic  acid.  For 
most  purposes  this  dilution  is  evidently  a  great  mistake,  for  the  concen- 
trated solution  is  itself  none  too  strong,  and  would  apparently  do  much 
better  if  it  were  much  stronger. 

90.  Picro-sulphuris  Acid  (KLEINENBEBG,  Quart.  Journ.  Mie.  Sci., 
April,  1879,  p.  208 ;  MAYER,  Journ.  Roy.  Mic.  Soc.  (N.  S.),  ii  (1882), 


68  CHAPTER    V. 

p.  867). — By  picro-sulphuric  acid,  without  any  qualifying  term,  I  under- 
stand a  fluid  made  (following  Mayer,  loc.  cit.)  as  follows  : — Distilled  water, 
100  vols. ;  sulphuric  acid,  2  vols. ;  picric  acid,  as  much  as  will  dissolve 
(this  will  be  about  0'25  per  cent.,  as  the  picric  acid  is  much  less  soluble  in 
sulphuric  acid  solution  than  in  water).  This  may  also,  in  any  case  in 
which  confusion  is  likely  to  arise,  be  called  "concentrated"  or  "undiluted 
picro-sulphuric  acid." 

By  "  liquid  of  Kleinenberg "  I  understand  a  mixture  suggested  by 
Kleinenberg  (loc.  cit.),  and  best  made  by  diluting  the  concentrated  picro- 
sulphuric  acid  prepared  as  above  with  three  times  its  volume  of  water. 

Of  these  two  formulis  the  one  formerly  most  employed  is  that  given  by 
Kleinenberg — the  dilute  mixture ;  undiluted  picro-sulphuric  acid  being 
reserved  for  objects  requiring  special  treatment,  chiefly  Arthropods.  I 
hold  that  Kleinenberg's  solution  is  much  weaker  than  is  desirable  in  the 
majority  of  cases,  and  should  be  reserved  for  special  cases,  such,  perhaps,  as 
that  for  which  it  was  originally  proposed,  the  embryology  of  the  earth- 
worm ;  the  concentrated  solution  being  generally  preferable.  This  particu- 
larly applies  to  marine  organisms. 

Wash  out  with  successive  alcohols,  beginning  with  70  per  cent.,  never 
with  water. 

Warm  alcohol  extracts  the  acid  much  more  quickly  than  cold,  without 
which  weeks  may  be  required  to  fully  remove  the  acid  from  chitinous 
structures. 

This  liquid,  once  the  classical  fixative,  is  now  almost  entirely  abandoned, 
I  think  rightly,  as  its  fixing  qualities  are  at  the  best  only  second-rate. 
For  Arthropoda  it  may  still  be  useful,  on  account  of  its  great  power  of 
penetrating  chitin.  For  a  fuller  account  see  previous  editions. 


91.  Picro-nitric  Acid  (MAYER,  Mitth.  Zool.  Stat.'Neapel,  1881,  p.  5  ; 
Journ.  Roy.  Mic.  Soc.  (N.S.),  ii,  1882,  p.  868).— 

Water 100  vols. 

Nitric  acid  (of  25  per  cent.  N205)  .         .         .         5     „ 

Picric  acid,  as  much  as  will  dissolve. 

The  properties  of  this  fluid  are  very  similar  to  those  of  picro-sulphuric 
acid,  with  the  advantage  of  avoiding  the  formation  of  gypsum  crystals,  and 
the  disadvantage  that  it  is  much  more  difficult  to  soak  out  of  the  tissues. 
The  process  of  .Telinek,  §  89,  may  be  useful  here.  Mayer  states  that  with 
eggs  containing  a  large  amount  of  yolk  material,  like  those  of  Palinurus, 
it  gives  better  results  than  nitric,  picric,  or  picro-sulphuric  acid.  I  myself 
consider  it  distinctly  superior  to  picro-sulphuric. 


02.  Picro-hydrochloric  Acid  (MAYER,  ibid.).— 

Water 100  vols. 

Hydrochloric  acid  (of  25  per  cent.  HC1)         .         8     ,, 
Picric  acid,  as  much  as  will  dissolve. 

The  properties  of  this  fluid  are  similar  to  those  of  picro-nitric  acid. 


FIXING    AND    HARDENING    AGENTS.  69 

93    Picro-chromic  Acid  (FoL,  Lehrb.,  p.  100).— 

Picric  acid,  sol.  sat.  in  water    .         .         .         .10  vols. 
1  per  cent,  chromic  acid  solution      .  .     25     „ 

Water 65     „ 

At  the  instant  of  using  you  may  add  O'OOo  of  osmic  acid,  which  makes 
the  action  more  energetic. 

I  have  seen  Fol's  formula,  with  the  addition  of  a  trace  of  acetic  acid, 
quoted  as  "  liquid  of  Haensel  " — I  know  not  with  what  justification. 

94.  Picro-nitro-chromic  Acid  (RAWITZ,  Leitfadtn  f.  liistol.  Unter- 
suchungen,  1895,  p.  24). — One  part  of    picro-nitric  acid,  and  four  parts 

1  per  cent,  chromic  acid.     Wash  out  in  70  per  cent,  alcohol. 

95.  Picro-osmic  Acid.— FLEMMING  (Zells.  Kern  u.  Zelltli.,  p.  381)  has 
experimented  with  mixtures  made  by  substituting  picric  for  chromic  acid  in 
the  chromo-osmic  mixtures  (ante,  §§  46  and  4,7).     The  results,  he  says,  are 
identical  so  far  as  regards  the  fixation  (of  nuclei) ;  but  staining  is  rendered 
more  difficult. 

0.  VOM  RATH  (Anat.  Anz  ,  xi,  1895,  p.  289)  adds  to  200  c.c.  of  saturated 
aqueous  solution  of  picric  acid,  12  c.c.  of  2  per  cent,  solution  of  osmic  acid, 
and  2  c.c.  of  glacial  acetic  acid. 

Flemming  may  be  right  as  regards  the  nuclei  ;  but  the  fixation  of  cyto- 
plasm is  in  my  preparations  decidedly  inferior. 

96.  Picro-nitro-osmic   Acid    (RAWITZ,   Leitfaden,   p.   24).  —  Picro- 
nitric  acid,  6  vols. ;  2  per  cent,  osmic  acid.,  1  vol.     Fix  for  £  to  3  hours. 

nsfer  direct  to  70  per  cent,  alcohol.     The  mixture  keeps  well. 

'  97.  Picro-platinic  and  Picro-platin-osmic  Mixtures. — 0.  TOM 
RATH  (Zoc.  cit.,  pp.  282,  285)  makes  a  picro-platinic  mixture  with  200  c.c. 
saturated  aqueous  solution  of  picric  acid,  1  g.  of  platinic  chloride  (dissolved 
in  10  c.c.  of  water),  and  2  c.c.  of  glacial  acetic  acid. 

The  picro-platin-osmic  mixture,  which  is,  in  my  opinion, 
much  superior,  is  made  by  adding  to  the  foregoing  25  c.c.  of 

2  per  cent,  osmic  acid. 

98.  Picric  Alcohol  (GAGE,  Proc.  Amer.  Soc.  Micr.,  1890,  p.  120; 
Jonrn.  Roy.  Mic.  Soc.,  1891,  p.  418). — Alcohol  (95  per  cent.),  250  parts; 
water,  250 parts;  picric  acid,  1  part. 


Other  Fixing  and  Hardening  Agents. 

99.  Alcohol. — For  fixing  it  is  generally  held  that  only  two 
grades  of  alcohol  should  be  employed — very  weak  alcohol  on 
the  one  hand,  and  absolute  alcohol  on  the  other  hand. 


70  CHAPTER    V. 

Absolute  alcohol  is  held  to  rank  as  a  fixing  agent  because  it 
is  said  to  kill  and  harden  with  such  rapidity  that  structures 
have  not  time  to  get  deformed  in  the  process  by  the  energetic 
dehydration  that  unavoidably  takes  place.  Dilute  alcohol  is 
held  to  rank  as  a  fixing  agent  in  virtue  of  being  of  such  a 
strength  as  to  possess  a  sufficiently  energetic  coagulating 
action  and  yet  contain  enough  water  to  have  but  a  feeble 
and  innocuous  dehydrating  action.  The  intermediate  grades 
do  not  realise  these  conditions,  and  therefore  should  not  be 
employed  alone  for  fixing.  But  they  may  be  very  useful  in 
combination  with  other  fixing  agents  (such  as  corrosive  sub- 
limate or  nitric  acid)  by  greatly  enhancing  their  penetrating 
power  ;  70  per  cent,  is  a  good  grade  for  this  purpose. 

A  recent  writer,  however,  TELLYESNICZKY  (Arch.  f.  Mik. 
Anat.,  lii,  2,  1898,  p.  219),  disagrees  with  the  doctrine  of  the 
supposed  importance  of  the  grades  of  alcohol  used  for  fixing, 
finding  them  all  equally  bad.  He  finds  no  difference  at  all 
between  the  action  of  absolute  alcohol  and  that  of  96  per 
cent,  or  that  of  70  per  cent.  They  all  cause  a  remarkable 
amount  of  shrinkage,  and  probably  a  notable  amount  of  solu- 
tion of  cell-constituents. 

A  point  that  is  not  sufficiently  attended  to  is  the  chemical  purity  of  the 
alcohol,  which  has  its  importance  in  respect  of  the  preservation  of  delicate 
.histological  detail  and  the  application  of  delicate  stains.  Pure  alcohol 
ought  to  leave  only  the  very  slightest  solid  residue  on  evaporation ; 
rubbed  between  the  hands  it  ought  not  to  leave  a  strong  odour  behind  after 
evaporation  ;  and  ought  to  show  neither  an  acid  nor  an  alkaline  reaction 
(this  latter  much  more  commonly  occurs  than  is  imagined).  It  should  be 
tested  with  very  sensitive  litmus  paper,  which  it  is  well  to  leave  in  it  for 
some  hours.  See  hereon  MAYER,  in  Mitt.  Zool.  Stat.  Neapel,  x,  1891,  p.  180, 
where  is  given  a  test  for  alkalinity: — a  solution  of  1  gramme  each  of  hae- 
matein  and  chloride  of  aluminium  in  100  c.c.  of  alcohol,  added  in  the 
proportion  of  1  : 100  to  the  alcohol  to  be  tested,  ought  not  to  be  precipitated 
in  twenty-four  hours. 

For  fixing,  alcohol  is  a  very  third-class  reagent,  only  to  be 
used  where  better  ones  cannot  be  conveniently  employed. 
For  hardening  it  is  a  very  important  one.  When  used  alone, 
it  is  indeed  inferior  as  a  hardening  agent  to  most  of  the 
reagents  discussed  above ;  but  when  judiciously  employed  to 
complete  the  action  of  a  good  fixing  agent,  it  renders  most 
valuable  services.  90  to  95  per  cent,  is  the  most  generally 
useful  strength.  Weaker  alcohol,  down  to  70  per  cent.,  is 


FIXING    AND    HAKDENING    AGENTS.  71 

often  indicated.  Absolute  alcohol  is  seldom  advisable.  You 
ought  to  begin  with  weak,  and  proceed  gradually  to  stronger, 
alcohol.  Large  quantities  of  alcohol  should  be  taken.  The 
alcohol  should  be  frequently  changed,  or  the  tissue  should 
be  suspended  near  the  top  of  the  alcohol,  in  order  to  have 
the  tissue  constantly  surrounded  with  pure  spirit  (the  water 
and  colloid  matters  extracted  from  the  tissue  falling  to  the 
bottom  of  the  vessel).  Many  weeks  may  be  necessary  for 
hardening  large  specimens.  Small  pieces  of  permeable 
tissue,  such  as  mucous  membrane,  may  be  sufficiently 
hardened  in  twenty-four  hours. 

100.  One-third  Alcohol. — The  grade  of  weak  alcohol  that  is 
generally  held  to  be  most  useful  for  fixing  is  one-third 
alcohol,  or  RANVIEK'S  ALCOHOL,  known  in  France  as  "  Alcool 
au  tiers,"  which  is  the  name  given  to  it  by  Ranvier  himself  ; 
in  Germany  as  "  .Drittelalcohol "  or  "  Ranviersche  alcohol 
dilutus  ;  "  in  Italy  as  "  alcool  al  terzo."  It  consists  of  two 
parttf  of  water  and  one  part  of  alcohol  of  90  per  cent,  (and  not 
of  absolute  alcohol,  as  was  stated  by  an  oversight  in  the  first 
edition — an  error  which  I  have  seen  copied  in  more  than 
one  place).  See  the  Traits  Technique  of  Ranvier,  p.  24 J,  et 
passim. 

According  to  Ranvier,  care  should  be  taken  that  the 
alcohol  is  of  the  strength  specified,  as  the  effects  of  this  re- 
agent depend,  he  states,  to  a  remarkable  degree  on  its 
strength. 

Objects  may  be  left  for  twenty-four  hours  in  this  alcohol ; 
not  more,  unless  there  be  no  reason  for  avoiding  maceration, 
which  will  generally  occur  after  that  time.  You  may  con- 
veniently stain  with  picro-carmine,  alum-carmine,  or  methyl 
green 

This  reagent  is  a  very  mild  fixative.  Its  hardening  action 
is  so  slight  that  it  is  not  at  all  indicated  for  the  fixing  of 
objects  that  are  intended  to  be  sectioned.  Its  chief  use  is 
for  extemporaneous  and  dissociation  preparations. 

101.  Absolute  Alcohol. — This  is  sometimes  valuable  on 
account  of  its  great  penetrating  power,  being,  indeed,  one  of 
the  most  penetrating  of  known  fixing  agents.  Mayer  finds 
that  boiling  absolute  alcohol  is  often  the  only  means  of  killing 


72  CHAPTER   V. 

certain  Arthropoda  rapidly  enough  to  avoid  maceration 
brought  about  by  the  slowness  of:  penetration  of  common 
cold  alcohol  (especially  in  the  case  of  Tracheata). 

It  is  important  to  employ  for  fixing  a  very  large  propor- 
tion of  alcohol.  Alum-carmine  is  a  good  stain  for  small 
specimens  so  fixed.  For  preservation,  the  object  should  be 
put  into  a  weaker  alcohol,  90  per  cent,  or  less. 

As  to  the  supposed  superiority  of  absolute  alcohol  over 
ordinary  strong  alcohol,  see  §  99  ;  and  amongst  authors 
upholding  its  superiority,  see  besides  RANVII-K,  MAYEU  (Mitth. 
Zool.  Stat.  Neapel,  ii,  1880,  p.  7)  ;  BRUEL  (Zool.  Jahrb.tAbth. 
Morph.,  Bd.  x,  1897,  p.  569)  ;  also  V^AN  REES  (ibid..,  Bd.  iii, 
1888,  p.  10). 

Absolute  alcohol  is  found  in  commerce.  It  is  a  product  that  it  is  almost 
impossible  to  preserve  in  use,  on  account  of  the  rapidity  with  which  it 
hydrates  on  exposure  to  air.  Fol  recommends  that  a  little  quicklime  be 
kept  in  it.  This  absorbs  part  at  least  of  the  moisture  drawn  by  the  alcohol 
from  the  air,  and  has  the  further  advantage  of  neutralising  the  acid  that  is 
frequently  present  in  commercial  alcohol. 

Another  plan  that  I  have  seen  recommended  is  to  suspend  strips  of  gelatin 
in  it.  It  is  stated  that  by  this  means  ordinary  alcohol  may  be  rendered 
absolute.  But  then  it  is  probably  also  rendered  very  acid  thereby. 

Ranvier  adopts  the  following  plan  for  preparing  an  alcohol  absolute 
enough  for  all  practical  purposes.  Strong  (95  per  cent.)  alcohol  is  treated 
with  calcined  cupric  sulphate,  with  which  it  is  shaken  up  and  allowed  to 
remain  for  a  day  or  two.  It  is  then  decanted  and  treated  with  fresh  cupric 
sulphate,  and  the  operation  is  repeated  until  the  fresh  cupric  sulphate  no 
longer  becomes  conspicuously  blue  on  contact  with  the  alcohol  ;  or  until,  on 
a  drop  of  the  alcohol  being  mixed  with  a  drop  of  turpentine,  no  particles  of 
water  can  be  seen  in  it  under  the  microscope.  The  cupric  sulphate  is  pre- 
pared by  calcining  common  blue  vitriol  in  a  porcelain  capsule  over  a  spirit 
lamp  or  gas  burner  until  it  becomes  white,  and  then  reducing  it  to  powder 
(see  Proc.  Acad.  Nat.  Sci.  Philad.,  1884,  p.  27  ;  Science  Record,  ii,  1884, 
p.  65 ;  Journ.  Roy.  Hie.  Soc.  (N.  S.),  iv,  1884,  pp.  322  and 


102.  Hydrochloric  Acid  Alcohol  (PAUL  MAYEE,  Mitth.  Zool.  Stat. 
Neapel,  ii,  1881,  p.  7). — To  97  vols.  of  90  per  cent,  alcohol,  in  which  is 
dissolved  a  small  quantity  of  picric  acid,  add  3  vols.  pure  hydrochloric  acid. 
Leave  the  specimens  in  the  mixture  only  just  long  enough  to  ensure  that 
they  are  thoroughly  penetrated  by  it.  Wash  out  with  90  per  cent,  alcohol, 
the  disappearance  of  the  yellow  stain  of  the  picric  acid  being  a  sign  that  all 
the  acid  is  removed. 

The  use  of  this  mixture  is  for  the  preparation  of  coarse  objects  it  is  in- 
tended to  preserve  in  alcohol.  The  object  of  the  acid  is  to  prevent  both 
that  glueing  together  of  organs  by  the  peri  visceral  liquid,  which  is  often 


FIXING    AND    HARDENING  AGENTS.  73 

brought  about  by  the  coagulating  action  of  pure  alcohol,  and  the  precipita- 
tion on  the  surface  of  organs  of  the  salts  contained  in  sea-water,  which  is  a 
hindrance  not  only  to  the  penetration  of  the  alcohol,  but  also  to  subsequent 
staining. 

Acid  alcohol  as  above  prepared  loses  its  original  qualities  after  standing 
some  time,  as  chloride  of  ethyl  is  gradually  formed  at  the  expense  of  the 
acid.  Seventy  per  cent,  alcohol  may  be  taken  instead  of  90  per  cent,  for 
making  the  mixture,  but  is  not  quite  so  good. 

Lo  BIANCO  (Mitth.  Zool.  Stat.  Neapel,  ix,  1890,  p. 443) takes  50  percent, 
alcohol  with  5  per  cent,  of  hydrochloric  acid. 

103.  Pyridin. — Pyridin  has  been  recommended  as  a  hardening  agent 
(by  A.  DE  SOUZA).  It  is  said  to  harden,  dehydrate,  and  clear  tissues  at  the 
same  time.  They  may  be  stained  after  hardening  by  anilin  dyes  dissolved 
in  the  pyridin,  or  passed  through  water  and  stained  by  the  usual  processes. 
It  is  said  to  harden  quickly,  and  to  give  particularly  good  results  with  brain. 
See  Comptes  Rendus  hebd.  de  la  Soc.  lie  Biologic,  8  ser.,  t.  iv,  No.  35, 
p.  622 ;  Zeit.f.  wise.  Mik.,  v,  i,  1888,  p.  65 ;  Journ.  ttoy.  Mic.  Soc.,  1888, 
p.  1054. 

104,  Formaldehyde  (Formol,  Formalin,  Formalose;. — Formal- 
dehyde is  the  chemical  name  of  the  gaseous  compound  HCOH, 
obtained  by  the  oxidation  of  methyl-alcohol.  "  Formalin  "  is 
the  commercial  name  given  by  SCH  KRING  &  Co.  to  a  40  per  cent, 
solution  of  this  substance  in  water.  "  Formol "  is  the  com- 
mercial name  given  to  the  same  solution  by  MKISTKU,  Lucius 
&  BKUNING.  And  "  Formalose  "  is  the  name  for  ihe  same 
solution  adopted  by  an  American  firm.  (These  solutions  may 
now  be  obtained  from  dealers  in  photographic  chemicals.) 
As  I  have  before  pointed  out  (Anat.  Anz.,  xi,  8,  1895,  p.  255), 
the  Already  extensive  literature  which  treats  of  the  anatomical 
uses  of  formaldehyde  is  much  confused  by  inaccurate  use  of 
these  terms  ;  many  writers  use  them  indiscriminately.  It  is 
frequently  impossible  to  discover  from  the  statements  of  an 
author  whether  he  means  such  or  such  a  percentage  of  formal- 
dehyde, or  such  or  such  a  percentage  of  the  commercial  40  per 
cent,  solution  employed  by  him,  the  one  being  of  course  two 
and  a  half  times  stronger  than  the  other.  All  that  can  be 
said  is,  that  the  majority  of  authors  seem  to  quote  in 
percentages  of  the  commercial  solutions.  I  think  it  must  be 
admitted  that  the  proper  way  of  stating  the  strengths  of  these 
solutions  is  either  to  state  them  in  terms  of  formaldehyde, 
and  say  so,  or  to  say  "formol,  or  formalin,  diluted  with  so 
many  volumes  of  water."  The  present  confusion  is  most 
inconvenient. 


74  CHAPTER  V. 

Solutions  of  formaldehyde  are  said  to  sometimes  decompose 
partially  or  entirely,  with  formation  of  a  white  deposit  of 
paraformaldehyde.  FISH  says  that  to  avoid  this  the  solutions 
should  be  kept  in  darkened  bottles  in  the  cool.  The  vapour 
of  formaldehyde  has  a  very  irritating  action  on  the  conjunctiva 
and  mucous  membranes,  but  the  effect  is  transitory,  not  so 
injurious  as  that  of  osmic  acid.  It  is  well  not  to  soil  the 
fingers  with  the  solutions,  as  formaldehyde  hardens  the 
living  skin  very  rapidly. 

It  was  discovered  independently  by  F.  BLUM  (Zeit.f.  wiss. 
Mik.,  x,  3,  1893,  p.  314)  and  by  HERMANN  (Anai.  Anz.,  ix,  4, 
1893,  p.  112)  that  formaldehyde  possesses  certain  hardening 
and  preservative  properties. 

BLOM  employed  formol  diluted  with  ten  volumes  of  water 
(containing  rather  less  than  4  per  cent,  of  formaldehyde). 
He  found  this  solution  to  penetrate  rapidly,  and  to  harden 
voluminous  organs  such  as  liver,  kidney,  brain,  more  rapidly 
than  alcohol,  and  that  sections  were  well  preserved  and  sus- 
ceptible of  good  staining. 

HERMANN  used  a  solution  containing  0'5  to  1  per  cent,  of 
"  formalin  "  (the  context  shows  that  1  per  cent,  of  formalde- 
hyde is  what  is  meant,  the  solution  being  made  by  diluting 
Sobering' s  formalin  with  forty  volumes  of  water).  He  found 
it  harden  very  rapidly,  with  the  remarkable  result  that  the 
hardened  organs  preserve  approximately  the  traiisparency  of 
life,  and  that  pigment*  are  not  discoloured.  Since  that  time 
formaldehyde  has  been  largely  used — in  some  cases  misused — 
for  the  preparation  and  preservation  of  must!  a  in  specimens, 
for  which  purpose  it  is  in  some  respects  superior  to  alcohol 
(for  the  employment  of  formaldehyde  in  museum  work,  see 
BLUM,  Zool.  Anz.,  xvi,  1893,  p.  450,  and  Verh.  An  at.  Ge#.,  8 
Yers.,  1895;  p.  236  ;  KAISEKLING,  Arch.  path.  Anat.,  Bd.  cxlvii, 
1897,  p.  396;  MELNIKOFF-RASVEDENKOFF,  Compt.Rend.,  t.  cxxiv, 
1897,  p.  238).  Signs  are,  however,  not  wanting  that  it  is  by  no 
means  the  elixir  that  has  been  supposed,  and  that  it  is  a 
great  mistake  to  imagine  that  it  can  take  the  place  of  alcohol 
as  a  definitive  preservative  of  anatomical  or  museum  specimens. 

It  was  said  above  that  formaldehyde  possesses  certain 
hardening  and  preservative  qualities,  the  limitation  intended 
being  that  it  does  not  harden  and  preserve  everything.  It 
hardens  gelatin,  for  instance,  and  certain  albuminoids  ;  but 


FIXING    AND    HARDENING   AGENTS.  75 

others,  on  the  contrary,  are  not  hardened  by  it,  but  rendered, 
011  the  contrary,  more  soluble  than  they  are  naturally.  For 
some  theoretical  considerations  concerning  its  action  on 
tissues,  see  F.  BLUM,  in  Anat.  Anz.,  xi,  1896,  p.  718; 
BENEDECENTJ,  in  Arch.  Anat.  u.  Phys,.  ,Abth.  1897,  p.  219; 
GEROTA,  in  Intern.  Monatschr.  Anat.  u.  Phys.,  xiii,  8,  1896, 
p.  108;  Zeit.f.  u-iss.  Mik.,  xiii,  3,  p.  311. 

On  account  of  its  hardening  properties  it  has  been  used 
as  a  fixing  agent.  On  account  of  the  confusion  in  termi- 
nology above  referred  to,  it  is  not  at  present  possible  to  give 
precise  instructions  as  to  the  strengths  that  have  been 
employed  by  the  different  authors  for  this  purpose.  All 
that  can  be  said  is  that  they  will  almost  certainly  be  bound 
to  lie  between  the  limits  of  those  indicated  by  BLDM  and 
HERMANN,  that  is  to  say  between  0*5  per  cent,  and  4  per 
cent,  if  the  formaldehyde  be  used  pure.  Only  one  writer 
(HoYER,  jun.,  Anat.  Anz.,  ix,  1894,  Erganzungsheft,  p.  236; 
Zeit.  f.  wiss.  Mik.,  xii,  1,  1895,  p.  28)  appears  to  have  used 
concentrated  solutions.  He  states  that  with  such  solutions 
tissues  are  better  preserved  than  with  weak  ones,  even 
better  preserved  than  with  corrosive  sublimate. 

There  is  certainly  some  mistake  here.  I  find  that  pre- 
parations fixed  in  13'H  per  cent,  formaldehyde  (formol  with 
two  volumes  of  water)  have  the  cells  enormously  over-fixed, 
and  presenting  the  homogeneous  aspect  of  osmicated  cells. 
Experimenting  further  with  weak  solutions  containing  from 
2  per  cent,  to  4  per  cent,  of  formaldehyde,  I  have  found 
that  like  the  stronger  solution  mentioned  above,  these  too 
give  a  homogeneous,  colloid  appearance  to  protoplasm,  and 
have  at  the  same  time  a  marked  swelling  and  vacuolating 
action.  With  the  2  per  cent,  solution  the  vacuolation  is 
enormous.  I  have  concluded  that  used  pure  formaldehyde 
is  not  at  all  suitable  as  a  fixing  agent  for  cytological  work, 
and  should  not  be  employed  for  that  purpose,  and  I 
certainly  should  not  think  of  using  it  myself,  even  for 
general  morphological  work. 

Some  formulae  for  formaldehyde  mixtures  for  fixing  given 
in  the  last  edition,  with  which  I  had  had  fair  results,  are 
suppressed,  as  I  now  think  that  where  the  results  were  good, 
it  may  have  been  in  spite  of  the  formaldehyde,  and  not  on 
account  of  it.  See,  however,  the  alcoholic  mixtures  of 


76  CHAPTER    V. 

LAVDOWSKY,  last  edition ;  or  the  picroformol  of  BOUIN 
(Phenomeiiex  cytologiqut's  anormaux  </a?i.s  V hittogenese ,  etc., 
Nancy,  1897)  (30  parts  picric  acid,  sol.  sat.,  10  parts  formol, 
2  parts  acetic  acid),  or  the  similar  one  of  GKAF  (Jonrii.  Roy. 
Mic.  8oc.t  1898,  p.  492),  the  mixture  of  ORTH  (10  parts 
formol  to  100  of  solution  of  Muller  [it  must  be  freshly  pre- 
pared, as  it  will  only  keep  a  few  days],  Berlin,  klin. 
Wocheitschr.,  1896,  No.  13;  Zeit.  f.  iciss.  Mik.,  xiii,  3,  p. 
316),  and  the  mixtures  quoted  under  "  Neurological  Method*  " 
in  Part  II. 

It  should  be  noted  by  those  who  desire  to  experiment 
with  such  mixtures,  that  formaldehyde  is  a  powerful  reducing 
agent,  and  therefore  incompatible  with  such  reagents  as 
chromic  acid  or  osmic  acid  and  the  like,  which  it  very 
rapidly  decomposes. 

To  sum  up,  I  feel  convinced  that  neither  strong  nor  weak 
should  formaldehyde  be  employed  pure  for  fixing ;  and  that 
it  has  not  been  shown  to  be  of  any  real  utility  in  fixing 
mixtures,  so  long  as  faithful  histological  fixation  is  aimed  at. 
But,  of  course,  that  does  not  prejudge  the  question  as  to 
whether  it  may  not  render  services  in  cases  in  which  faithful 
histological  fixation  is  only  a  secondary  point,  as  in  many 
pathological  researches  ;  nor  does  it  imply  that  it  may  not 
be  useful  for  the  purpose  of  hardening  sensu  stricto  tissues 
that  have  previously  been  duly  fixed  by  some  other  agent. 

Considered  as  a  hardening  agent  sensu  strict o  the  most 
important  use  of  formaldehyde  is  for  hardening  nervous 
tissue,  for  which  it  is  now  much  used,  perhaps  with  better 
justification  than  for  most  other  purposes.  For  this  see 
Part  II. 

HERMANN  (loc.  cit.,  supra)  found  that  such  a  large  organ  as 
a  calf's  heart  was  entirely  hardened  by  a  0*5  to  1  per  cent, 
in  twelve  to  twenty-four  hours.  Entire  eye*  are  so  hardened 
in  the  I  per  cent,  solution  in  twenty-four  hours  that  they 
may  be  cut  in  two  with  a  sharp  knife,  like  an  apple.  Her- 
mann found  this  disadvantage,  that  tissues  hardened  in 
formaldehyde  solution  suffer  when  they  are  put  into  alcohol 
for  the  purpose  of  dehydration.  The  paper  in  question  con- 
tains interesting  observations  on  the  property  formaldehyde 
has  of  preserving  the  natural  colours  and  transparent  and 
lifelike  aspect  of  tissues. 


FIXING   AND    HARDENING    AGENTS.  77 

BLUM  (Anat.  Anz.,  ix,  1894,  p.  229),  recapitulating,  says- 
that  very  voluminous  pieces  of  material  are  hardened  quickly 
and  without  shrinkage.  The  tissues  stain  well.  Cells,  he 
sava;,  and  nuclei  preserve  their  forms  ;  karyokinetic  figures 
are  fixed.  Mucin  is  not  precipitated,  but  remains  trans- 
parent; fat  is  not  dissolved.  Micro-organisms  retain  their 
specific  staining  reactions. 

As  to  the  degree  and  hind  of  hardening  obtained  by  for- 
maldehyde the  authors  are  not  so  explicit  as  could  be  wished. 
As  far  as  I  can  see  myself,  the  hardening  obtained  is  gentle 
and  tough,  giving  an  elastic  and  not  a  brittle  consistency. 
It  varies  greatly  with  different  tissues. 

For  prolonged  hardening,   considerable  volumes  of  liquid 
should  be  taken,  and  the  liquid  should  be  renewed  from  time 
to    time  ;     for  the  formaldehyde  fixes  itself  on  the  tissues 
with  which  it  comes  in  contact,  deserting  the  solution,  whic 
thus  becomes  progressively  weaker. 

For  further  hints  concerning  hardening  with  formalde- 
hyde see  the  papers  of  F.  BI.UM  and  GEROTA  above  quoted.  I 
cannot  but  suspect  that,  both  for  nervous  system  and  other 
purposes,  it  is  now  being  used  with  much  more  enthusiasm 
than  critical  judgment. 

Formaldehyde,  being  a  powerful  reducing  agent,  may  be 
employed  for  the  reduction  of  gold  and  silver  impregnations. 
I  have  been  using  it  myself  for  reducing  gold  impregnations, 
and  up  to  the  present  like  it  better  than  any  other  agent  I 
have  tried  for  that  purpose. 

It  is  also  a  powerful  antiseptic,  and  may  be  found  very 
useful  for  effecting  the  preservation  of  staining  solutions,  with 
some  of  which  it  acts  as  a  mordant.  It  is  said  to  harden 
celloidin  as  well  as  gelatin,  and  to  be  useful  for  celloidin- 
imbedding  (BLUM,  Anat.  Anz.,  xi,  1896,  p.  724). 


CHAPTER    VI. 

DE-ALCOHOLISATiON    AND    CLEARING    AGENTS. 

105.  Introductory  Remarks. — De-alcoholisation  agents*  are 
liquids  employed  for  the  purpose  of  getting  rid  of  the  alcohol 
in  which  preparations  are  generally  preserved,  and  facili- 
tating the  penetration  of  the  paraffin  used  for  imbedding,  or 
the  balsam  or  other  resinous  medium  in  which  preparations 
are,  in  most  cases,  finally  mounted.  Hence  all  of  them 
must  be  capable  of  expelling  alcohol  from  tissues,  and  must 
be  at  the  same  time  solvents  of  Canada  balsam  and  the  other 
resinous  mounting  media.  The  majority  of  them  are  essen- 
tial oils. 

Clearing  agents  are  liquids  whose  function  it  is  to  make 
microscopic  preparations  ^transparent  by  penetrating  amongst 
the  highly  refracting  elements  of  which  the  tissues  are  com- 
posed, the  clearing  liquids  themselves  having  an  index  of 
refraction  superior,  or  equal,  or,  at  all  events,  not  greatly 
inferior  to  that  of  the  tissues  to  be  cleared.  Hence  all 
clearing  agents  are  liquids  of  high  index  of  refraction. 

The  majority  of  de-alcoholisation  agents  being  also  liquids 
of  high  refraction,  it  follows  that  they  serve  at  the  same  time 
for  de-alcoholisation  and  for  clearing ;  and  in  consequence 
it  has  come  about  that  de-alcoholisation  agents  are  generally 
spoken  of  as  clearing  agents.  (The  expression  "  de-alcoholi- 
sation agents  "  is  here  used  for  the  first  time  as  a  correlative 
of  the  "Vormedien"  of  APATHY  (Mikrotechnic) ,  and  the 
"  Vorharze  "  of  the  Grundzuge,  due  to  MAYER  ;  in  previous 
editions  clearing  agents  and  de-alcoholisation  agents  were 
indiscriminately  spoken  of  as  clearing  agents.)  But  that 
practice  is  not  strictly  correct,  for  not  all  clearing  agents  are 
solvents  of  tlie  resins,  and  not  all  de-alcoholisation  agents 
can  serve  as  clearers.  For  instance,  glycerine  is  a  clearing 
agent,  but  cannot  be  used  to  prepare  objects  for  paraffin  or 


DE-ALCOHOLISATION    AND    CLEARING    AGENTS. 

for  balsam,  because  it  is  not  miscible  with  either  of  them. 
And  chloroform  is  an  admirable  de-alcoholisation  agent  and 
admirable  precursor  of  paraffin  or  balsam,  but  can  hardly  be 
utilised  as  a  clearer — i.  e.  for  the  purpose  of  obtaining  the 
transparency  required  for  examination — on  account  of  its 
volatility,  which  precludes  its  use  as  an  examination  medium. 
1  shall,  however,  still  in  many  cases  continue  to  use  the  term 
*'  clearing "  to  signify  "  de-alcoholising,"  for  the  sake  of 
brevity. 

Of  course  clearing  media  can  serve  as  Examination  Media, 
if  not  too  volatile. 

106.  The  Practice  of  De-alcoholisation  or  Clearing.— The 
old  plan  was  to  take  the  object  out  of  the  alcohol  and  float 
it  on  the  surface  of  the  de-alcoholising  or  clearing  medium 
in  a  watch-glass.  This  plan  was  faulty,  because  the  alcohol 
escapes  from  the  surface  of  the  object  into  the  air  quicker 
(in  most  instances)  than  the  de-alcoholising  or  clearing  agent 
can  get  into  it  ;  hence  the'object  must  shrink.  To  avoid  or 
lessen  this  cause  of  shrinkage,  the  operation  is  now  generally 
done  by  the  method  suggested  by  Mayer  and  Giesbrecht, 
which  consists  in  putting  the  clearing  medium  under  the 
alcohol  containing  the  object.  This  is  done  in  the  following 
manner.  Take  a  short  glass  tube,  and  put  into  it  enough 
alcohol  to  contain  the  objects  (a  watch-glass  will  often  do 
well,  but  a  tube  is  safer).  With  a  pipette  carefully  put 
under  the  alcohol  a  sufficient  quantity  of  clearing  medium 
(or  carefully  pour  the  alcohol  on  to  the  clearing  medium). 
Then  put  the  objects  into  the  alcohol.  They  will  sink  down 
to  the  level  of  separation  of  the  two  liquids  at  once  ;  and 
after  some  time  they  will  be  found  to  have  sunk  to  the 
bottom  of  the  clearing  medium.  They  should,  however,  not 
be  considered  to  be  perfectly  penetrated  by  the  clearing 
medium  until  the  wavy  refraction-lines  caused  by  the  mixture 
of  the  two  liquids  at  their  surface  have  ceased  to  form.  They 
may  then  be  removed  by  means  of  a  pipette,  or  the  super- 
natant alcohol  drawn  off  and  the  preparations  allowed  to 
remain  until  wanted. 

The  penetration  of  all  clearing  media  may  be  hastened  by 
using  them  warm. 

It   frequently  happens  that  the   essential   oil  with  which 


80  CHAPTER  VI. 

objects  are  being  treated  in  a  watch-glass  or  011  a  slide 
becomes  cloudy  after  a  short  time,  and  fails  to  clear  the 
tissues.  This  is  owing  to  a  combination  between  the  essen- 
tial oil  and  moisture,  derived,  I  think,  rather  from  the  air 
than  from  the  objects  themselves.  The  cloudiness  can 
usually  be  removed  by  warming  (as  pointed  out  by  HATCHETT 
JACKSON,  Zool.  Anzeig.,  1889,  p.  630),  but  this  remedy  is  not 
always  successful,  for  in  certain,  moist  states  of  the  atmo- 
sphere the  cloudiness  will  persist,  notwithstanding  continued 
warming.  It  is  for  this  reason  that  I  advise  that  clearing* 
be  done,  whenever  possible,  in  shallow,  well-corked  tubes, 
under  which  conditions  the  phenomenon  rarely  occurs.  In 
any  case,  be  careful  not  to  breathe  on  the  liquid. 

107.  Classification  of  De-alcoholisation  and  Clearing  Agents, — 

NEELSEN  and  SCHIEFFERDECKEK  (Arch.  f.  Anat.  u.  Phys., 
1882,  p.  206)  examined  a  large  series  of  ethereal  oils  (pre- 
pared by  Schimmel  and  Co.,  Leipzig),  with  the  object  of 
finding  a  not  too  expensive  substance  that  should  combine 
the  properties  of  clearing  quickly  alcohol  preparations,  not 
dissolving  out  anilin  colours,  clearing  celloidin  without  dis- 
solving it,  not  evaporating  too  quickly,  and  not  having  a  too 
disagreeable  smell. 

Of  these,  the  following  three  fulfil  the  conditions  and  can 
be  recommended: — Cedar-wood,  Origanum,  Sandal-wood. 

To  these  should  be  added  the  others  recommended  in  the 
following  paragraphs. 

See  also  the  paper  of  JORDAN,  Zeit.  f.  wisa.  Mik.,  xv,  i, 
1898,  p.  50,  which  has  special  reference  to  the  behaviour  of 
some  essential  oils  towards  celloidin. 

It  would  be  important  to  possess  a  complete  list  of  the  exact  indices  of 
refraction  of  the  substances  used  for  clearing.  I  have,  unfortunately,  not 
been  able  to  obtain  sufficient  information  of  a  trustworthy  nature  for  the 
compilation  of  such  a  list.  Cedar  oil  has  nearly  the  index  of  crown  glass 
(this  is  true  of  the  oil  in  the  thick  state  to  which  it  is  brought  by  exposure 
to  the  air,  not  of  the  new,  thin  oil,  which  is  less  highly  refractive),  it 
therefore  clears  to  about  the  same  extent  as  Canada  balsam.  Clove  oil  has 
a  much  higher  index,  and  therefore  clears  more  than  balsam;  cinnamon 
oil  higher  still.  Turpentine  and  bergamot  oil  have  much  lower  indices, 
and  therefore  clear  less. 

The  following  short  list,  extracted  from  BEHKENS'  Tabellen  zutn  Ge- 
brauch  bei  mikroskopischen  Arbeiten,  Braunschweig,  1892,  p.  42,  and 


DE-ALCOHOLTSATION    AND    CLEARING    AGENTS. 


81 


other  sources,  may  be  useful,  but  the  figures  must  be  accepted  with  some- 
caution,  on  account  of  the  variability  of  samples.  The  figures  given  for 
balsam  refer  evidently  to  the  resin  in  the  solid  state  and  not  to  the  solu- 
tions used  for  mounting,  which  are  certainly  much  lower  according  to  the- 
lower  index  of  the  solvent. 


Air 1-000 

Methyl  alcohol     .         .         .  1*323 

Distilled  water     .         .         .  1*336 

Sea  water     ....  1-343 

Absolute  alcohol  .         .         .  1*367 
Acetate  of  potash,  saturated 

aqueous  sol.  .  .  .  1*370 
Glycerin  with  an  equal  quan- 
tity of  water  .  .  .  1-397 
Glycerin,  Price's  .  .  .  1*460 
Oil  of  bergamot  .  .  .  1*464 
Paraffinum  liquidum  .  .  1*471 
Olive  oil  ....  1-473 
Oil  of  turpentine  .  .  1*473 
Glycerin,  "  concentrated  "  .  1'473 
Castor  oil  ....  1*490  j 
See  also  the  chapter  on 


Cedar-wood  oil,  not  thickened  1*510 
Crown  glass  .  .  ,  1*518 
Cedar-wood  oil,  thickened  .  1*520 
Oil  of  lemons  .  .  .  1*527 
Oil  of  cloves  .  .  .  1*533 
Canada  balsam  (solid) .  .  1*535 
Creasote  ....  1*538 
Carbolic  acid  .  .  .  1*549 
Oil  of  anise  seed  .  .  .  1'557 
Anilin  oil  .  .  .  .  1*580 
Oil  of  cinnamon  .  .  .  1*619 
Sulphide  of  carbon  .  .  1*630 
Tolu  balsam  .  .  .  1*640 
Monobromide  of  naphthalin  T66O 
Solution  of  sulphur  in  sul- 
phide of  carbon  .  .  1*750 
'  Examination  Media." 


108.  Choice    of    a   De-alcoholisation    or   Clearing   Agent. — 
Special   directions   for   clearing  are    given   when  necessary 
under  the  heads  of  the  different  organs  and  tissues.      It  will 
suffice  here  to  advise  the  beginner  to  keep  on  his  table  the 
following : — Oil  of  cedar,  for  general  use  and  for  preparing 
objects  for  imbedding  in  paraffin,  see  "Imbedding  Methods 
— Paraffin  ;  "   clove   oil,  for   making   minute    dissections  in 
cases    in  which  it    is    desirable   to   take   advantage  of  the 
property  of  that  essence  of  forming  very  convex  drops  on 
the  slide,  and  of   imparting  a  remarkable  brittleness  to  soft 
tissues,  and  for  much  work  with  safranin,  etc. ;  oil  of  bergamot 
which  will  clear  from   90  per  cent,  alcohol,  and  which  does 
not  extract  coal-tar  colours  ;  carbolic  acid,  for  rapidly  clear- 
ing very  imperfectly  dehydrated  objects. 

For  special  clearers  for  celloidin  sections  see  "  Collodion 
(Celloidin)  Imbedding  Methods." 

109.  Cedar    Oil    (NEELSEN  and  SCHIEPPERDECKER,  loc.  cit., 
§  107). — Thin,  colour  light  yellow  or  greenish,  odour  slight 
(of  cedar- wood),  evaporates  slowly,  is  not  changed  by  light, 
is    miscible   with    chloroform -balsam,   and   with    castor  oil. 

6 


'82  CHAPTER  VI. 

Clears  readily  tissues  in  95  per  cent,  alcohol  without  shrink- 
age ;  does  not  extract  anilin  colours.  Celloidin  sections  are 
cleared  in  five  to  six  hours. 

Cheap,  but  requires  an  inconvenient  length  of  time  for 
the  clearing  of  celloidin  sections. 

The  observer  should  be  careful  as  to  the  quality  of  the 
cedar  oil  he  obtains..  I  have  examined. the  clearing  properties 
of  a  sample  obtained  from  the  celebrated  firm  of  Rousseau, 
Paris.  •  This  sample  was  absolutely  colourless.  It  totally 
failed  to  clear  absolute  alcohol  objects  after  many  days. 

Cedar  oil  is  very  penetrating,  and  for  this  and  other 
reasons  is,  in  my  experience,  the  very  best  of  all  media  for 
preparing  objects  for  paraffin  imbedding.  I  find  it  to  be 
less  hurtful  to  cells  and  delicate  tissue-structures  than  any 
other  medium  known  to  me. 

110.  Clove  Oil. — Samples  of  clove  oil  of  very  different 
shades  of  colour  are  met  with  in  commerce.  It  is  frequently 
recommended  that  only  the  paler  sorts  should  be  employed 
in  histology.  A  word  of  explanation  is  here  necessary. 
Doubtless  it  is,  in  general,  best  to  use  a  pale  oil,  provided 
it  be  pure,  but  it  is  not  always  easy  to  obtain  a  light- 
coloured  oil  that  is  pure.  Clove  oil  passes  very  readily 
from  yellow  to  brown  with  age,  so  that  in  choosing  a  colour- 
less sample  you  run  great  risk  of  obtaining  an  adulterated 
sample,  for  clove  oil  is  one  of  the  most  adulterated  sub- 
stances in  commerce. 

Two  important  properties  of  clove  oil  should  be  noticed 
here.  It  does  not  easily  spread  itself  over  the  surface  of  a 
slide,  but  has  a  tendency  to  form  very  convex  drops.  This 
property  makes  it  a  very  convenient  medium  for  making 
minute  dissections  in.  The  second  property  I  wish  to  call 
attention  to  is  that  of  making  tissues  that  have  lain  in  it  for 
.some  time  very  brittle.  This  brittleness  is  'also  sometimes 
very  helpful  in  minute  dissections. 

These  qualities  may  be  counteracted  if  desired  by  mixing 
the  clove  oil  with  bergamot  oil. 

Clove  oil  has  a  high  index  of  refraction,  and,  clears  ob- 
jects more  than  balsam  mounting  media.  It  dissolves  cel- 
loidin (or  collodion),  and  therefore  should  not  be  used  for 
•clearing  sections  cut  in  that  medium,  without  special  pre- 


DE-ALCOHOLISATION   AND   CLEANING   AGENTS.  83 

Cautions.  I  consider  this  to  be  one  of  the  best  of  clearing 
agents,  and  valuable  on  account  of  the  properties  to  which 
attention  has  been  called  above.  New  clove  oil  washes  out 
anilin  colours  more  quickly  than  old.  It  is  well  to  possess 
trustworthy  samples  of  both  new  and  old  oil. 

111.  Cinnamon  Oil  (Erratim  "  Cannel  oil "  in  previous  editions) 
greatly  resembles  clove  oil,  but  is  in  general  thinner,  and  is  more  highly 
refractive.  An  excellent  medium,  which  I  particularly  recommend. 

112.  Oil  of  Bergamot  (SCHIEFFEKDECKER,  Arch.  Anat.  u. 
Phys.,  1882  [Anat.  Abth.],  p.  206).— This  oil  clears  95  per 
cent,  alcohol  preparations  and  celloidin  preparations  quickly, 
and  does  not  attack  anilin  colours.      I  think  that  this  is 
valuable  medium,  and  though  I  do  not  agree  with  Schieffer- 
decker  in  thinking  its  action  superior  to  oil  of  cloves,  I  think 
it  should  always  be  kept  at  hand. 

Bergamot  oil  is,  I  believe,  the  least  refractive  of  these 
essences,  having  a  lower  index  than  even  oil  of  turpentine. 

SUCHANNEK  (Zeit.  f.  wiss.  Mik.,  vii,  2,  1890,  p.  158)  says 
that  bleached,  colourless  bergamot  oil  will  not  take  up  much 
water,  whereas  a  green  oil  will  take  up  as  much  as  10  per 
cent. 

VAN  DEB  STRICHT  (Arch,  de  BioL,  xii,  1892,  p.  741)  says 
that  bergamot  oil  will,  with  time,  dissolve  out  the  fatty 
granules  of  certain  ova. 

113.  Oil  of  Origanum  (NEELSEN  and  SCHIEFFERDECKER,  Arch. 
Anat.  u.  Phys.,  1882,  p.  204).— Thin,   light   brown    colour, 
odour  not  too   strong,   agreeable,    does   not    evaporate    too 
quickly,  is  not  changed  by  light,  is  miscible  with  chloroform- 
balsam   and  with  castor  oil.      Ninety-five  per  cent,  alcohol 
preparations  are  cleared  quickly,  and  so  are    celloidin    sec- 
tions, without  solution  of  the  celloidin.      Anilin  colours  are 
somewhat  extracted. 

For  work  with  celloidin  sections  care  should  be  taken  to 
obtain  01.  Oriyani  Cretici  ("  Spanisches  Hopfenol "),  not  01. 
Orig.  Gallici  (v.  G-IESON  ;  see  Zeit.  f.  wiss.  Mile.,  iv,  4,  1887, 
p.  482).  Specimens  of  origanum  oil  vary  greatly  in  their 
action  on  celloidin  sections,  and  care  should  be  taken  to 
obtain  a  good  sample. 


84  CHAPTER   VI. 

SQUIRE,  in  his  Methods  and  Formulas,  etc.,  p.  81,  says  that 
origanum  oil  (meaning  the  commercial  product)  is  nothing 
but  oil  of  white  thyme  more , or  less  adulterated  (see  next  §)r 
and  that  the  product  sold  as  01.  Origani  Cretici  is  probably 
oil  of  marjoram. 

114.  Oil  of  Thyme.— FISH  (Proc.  Amer.  Hie.  Soc.,  1893  ; 
Zeit.f.  wiss.  Mik.,  xi,  4,  p.  503),  following  BDMPUS,  says  that 
for  most  of  the  purposes  for  which  origanum  oil  has  been 
recommended,,  oil  of  thyme  will  do  just  as  well  if  not  better. 
After  one  distillation  of  the  crude  oil  of  thyme  it  is  of  a  red- 
dish-brown colour,  and  is  called  the  red  oil  of  thyme  ;  when 
again  distilled  it  becomes  colourless,  and  is  distinguished  as 
the  white  oil.  The  red  oil  is  just  as  efficient  as  the  white  for 
clearing. 

Schimmeland  Co.,  in  their  Report  of  October,  1895,  p.  69, 
state  that  in  France  white  oil  of  thyme  is  adulterated  with 
oil  of  turpentine  to  the  extent  of  as  much  as  50  per  cent. 
(MAYER)  . 

115.  Sandal -wood   Oil   (NEELSEN   and    SCHIEFFERDECKEK,  ibid.). — 
Very  useful ;  but  its  high  price  is  prohibitive. 

116.  Oil  of  Cajeput. — This  oil  is,  I  believe,  frequently  used  as  a  clearer 
by  the  botanists.     I  have  used  it  myself  and  found  it  to  clear  well,  but  to- 
be  rather  thin.     CABNOY  AND  LEBEUN  (La  Cellule,  xiii,  1,  1897,  p.  71) 
have  found  it  useful  for  clearing  celloidin  sections.     It  dissolves  celloidin 
very  slowly  and  clears  without  shrinkage. 

117.  Oil  of  Turpentine. — Generally  used  for  treating  sections  that 
have  been  cut  in  paraffin,  as  it  has  the  property  of  dissolving  out  the 
paraffin  and  clearing  the  sections  at  the  same  time  ;  but  many  other  re- 
agents (see  §  139)   are  preferable  for  this  purpose.     If  used  for  alcohol 
objects  it  causes  considerable  shrinkage,  and  alters  the  structure  of  cells 
more  than  any  other  clearing  agent  known  to  me.      Turpentine  has,  I 
believe,  the  lowest  index  of  refraction   of  all  the  usual  clearing  agents 
except  bergamot  oil;  it  clears  objects  less  than  balsam. 

118.  Carbolic  Acid. — Best  used  in  concentrated  solution  in 
alcohol.  Clears  instantaneously,  even  very  watery  prepara- 
tions. This  is  a  very  good  medium,  but  it  is  better  avoided 
for  preparations  of  soft  parts  which  it  is  intended  to  mount 
in  balsam,  as  they  generally  shrink  by  exosmosis  when  placed 


DE-ALCOHOLISATION   AND    CLEARING   AGENTS.  85 

in  the  latter  medium.      It  is,  however,  a  good  medium  for 
celloidin  sections. 

119.  GAGE'S  Mixture  (Proc.  Amer.  Soc.  Micr.,  1890,  p.  120;  Journ. 
Roy.  Hie.  Soc.,  1891,  p.  418). — Carbolic  acid  crystals  melted,  40c.c. ;  oil  of 
turpentine,  60  c.c. 

120.  Creasote. — Much  the  same  properties  as  carbolic  acid. 
Beech-wood  creasote  is  the  sort  that  should  be  preferred  for 
many  purposes, — amongst  others,  for  clearing  celloidin 
sections,  for  which  it  is  a  very  good  medium. 

121.  Anilin  Oil. — This  is  a  rather  important  reagent  on  account  of  its 
ability  to  clear  excessively  watery  objects.  Common  anilin  oil  will  readily 
clear  sections  from  70  per  cent,  alcohol,  and  with  certain  precautions  (for 
which  see  the  paper  of  SFCHANNEK  quoted  below)  objects  may  be  cleared 
from  watery  media  without  the  intervention  of  alcohol  at  all.  This  pro- 
perty renders  anilin  valuable  in  certain  cases  as  a  penetrating  medium  for 
preparing  for  paraffin  imbedding.  For  ordinary  work  the  usual  commercial 
anilin  will  suffice;  and  it  is  immaterial  whether  it  be  colourless  or  have 
become  brown  through  oxidation.  For  difficult  work  it  is  well  to  use  a 
perfectly  anhydrous  oil.  For  directions  for  preparing  this  see  SUCHANNEK, 
eit.f.  wiss.  Mik.,  vii,  2,  1890,  p.  156,  or  the  third  edition  of  this  work. 
Anilin  is  chiefly  used  for  clearing  celloidin  sections,  and  is  sometimes 
found  very  valuable  for  this  purpose. 

122.  Xylol,  Benzol,  Toluol,  Chloroform. — Too  volatile  to  be 
recommendable  as  clearing  agents  in  which  it  is  desired  to 
examine  specimens,  but  very  useful  for  preparing  paraffin 
sections  for  balsam.  Of  the  three  first-mentioned  liquids, 
benzol  is  the  most  volatile,  then  toluol,  and  xylol  is  the  least 
volatile,  in  the  proportion  of  4 :  5  :  9  (SQUIRE,  Methods  and 
Formulas,  p.  20).  Chloroform  is  injurious  to  some  delicate 
stains,  but  is  in  other  respects  an  excellent  de-alcoholisation 
agent,  as  it  will  take  up  a  good  deal  of  water,  if  any  be  left 
in  the  preparations.  1  consider  it  too  volatile  for  use  before 
balsam.  Xylol  is  the  best  of  these  in  that  respect ;  but  it 
has  the  defect  of  mixing  very  slowly  with  alcohol.  I  now 
generally  bring  my  sections  from  alcohol  first  into  chloroform, 
to  remove  the  alcohol ;  then  into  xylol,  and  thence  into  the 
balsam. 

123.  Amyl  Alcohol.— JANSSENS  (La  Cellule,  xiv,  1,  1898,  p.  209) 
treats  cover-glass  preparations,  taken  from  95  per  cent,  alcohol,  with  amyl 


86  CHAPTER    VI. 

alcohol  before  mounting  in  damar  or  colophonium,  with  the  view,  if  I 
understand  rightly,  of  more  efficaciously  completing  the  dehydration  of  the- 
preparations.  I  do  not  understand  whether  he  mounts  direct  from  the 
arnyl  alcohol  or  passes  through  an  essence. 

123a.  Methyl  Salicylate.— This  is  recommended  by  GUEGUEN  (Comp. 
Rend.  Soc.  Biol.,  v,  1898,  p.  285 ;  Journ.  Roy.  Mic.  Soc.,  1899,  p.  456) 
both  as  a  de-alcoholisation  and  clearing  agent,  and  as  a  solvent  of  paraffin. 


CHAPTER  VII. 
IMBEDDING   METHODS — INTRODUCTION. 

124.  A  word  on  Microtomes. — It  is  no  part  of  the  purpose  of  this- 
work  to  discuss  instruments,  yet  a  word  on  this  subject  may  be  helpful  to 
the  student.  The  freezing  microtome  so  generally  employed  in  England, 
and  no  doubt  highly  useful  for  the  pathologist,  is  less  than  any  other 
form  adapted  to  the  wants  of  the  zoologist.  Very  thin  sections  can  be 
obtained  by  it  more  readily  than  with  any  other  microtome,  but  they  are  of 
little  use  when  obtained.  The  relations  of  the  parts  of  the  organs  are 
deranged  by  the  freezing  and  by  the  thawing,  and  the  aqueous  nature  of 
the  process  prevents  it  from  being  readily  applicable  to  the  mounting  of 
series  of  sections.  The  microtome  of  the  zoologist,  therefore,  must  be  an 
imbedding  microtome. 

Now  there  are  two  methods  of  imbedding  in  general  use — the  paraffin 
method  and  the  celloidin  method.  In  the  paraffin  method  the  object  is  cut 
dry,  with  the  knife  set  square  ;  whilst  in  the  celloidin  method  the  object  is 
usually  cut  wet,  and  in  a  softer  and  more  elastic  state  than  paraffin  objects, 
and  always  with  an  obliquely-set  knife.  It  so  happens  that  the  most  precise 
and  beautiful  microtomes  that  have  been  constructed  are  designed  in  view 
of  the  paraffin  method,  and  cannot  be  applied,  or  at  all  events  are  much  less 
adapted,  to  work  with  celloidin  objects.  A  thoroughly  equipped  laboratory 
should  therefore  possess  two  microtomes,  one  for  paraffin  work,  and  one  for 
celloidin  material,  or  other  material  that  has  to  be  cut  in  the  wet  way.  If 
the  anatomist  cannot  afford  two  instruments,  he  will  perhaps  do  well  not  ta 
choose  one  of  those  that  are  adapted  only  for  paraffin,  but  to  choose  an  all- 
round  instrument,  one  that  without  being  absolutely  of  the  highest  attain- 
able precision  in  paraffin  work  will  yet  give  sufficiently  good  results  in  that 
way,  and  will  also  cut  in  the  wet  way. 

Amongst  microtomes  fulfilling  these  conditions  various  forms  will  be 
found  almost  equally  convenient.  Zeiss  makes  a  good  one;  Schanze,  of 
Leipzig,  makes  a  good  one;  Reichart,  of  Vienna,  makes  a  good  one.  All 
these  are  relatively  cheap,  and,  being  at  the  same  time  perfectly  efficient 
for  easy  work,  may  be  recommended.  Amongst  more  precise  instruments 
of  this  class  I  particularly  recommend  the  THOMA  sliding  microtome,  as 
made,  in  several  sizes,  by  R.  Jung,  Mechaniker  in  Heidelberg.  For 
zoological  and  general  histological  work  I  recommend  the  medium  size 
N  .  tj,  with  the  newest  Naples  object-holder  and  newest  form  of  knife  and 
knife-holder. 


S8  CHAPTER   VII. 

This  instrument  is  described  in  Journ.  Roy.  Mic.  Soc.  (N.S.),  vol.  iii, 
p.  2^8  ;  the  new  Naples  object-holder  (which  I  consider'  essential  for  the 
zoologist)  is  described  and  figured  p.  915.  See  also  the  improvements 
described  op.  cit.,  1887,  p.  334,  and  the  latest  price  list  of  E.  Jung  (which 
may  be  obtained  from  Mr.  C.  Baker,  Optician,  244,  High  Holborn,  London, 
W.C.). 

The  BECZEK  microtome  is  also  very  much  to  be  recommended  (at  least  for 
paraffin  work  :  as  to  celloidin  I  cannot  say).  It  is  essentially  on  the  same 
principle  as  the  Thoma,  but  possesses  a  mechanical  arrangement  for  moving 
the  knife-carrier;  that  is,  the  knife-carrier  is  not  only  guided  by  a  mecha- 
nical arrangement,  as  in  the  Thoma  model,  but  is  put  in  motion  by  mecha- 
nism. This  is  frequently  an  advantage.  It  is  made  by  Aug.  Becker, 
•Gottingen.  Descriptions  of  two  forms  (Spengel  and  Schiefferdecker)  will 
"be  found  in  Journ.  Roy.  Mic.  Soc.,  1886,  pp.  884  and  1084.  The  Naples 
object-holder  can  be  fitted  to  the  Becker  microtome. 

The  instruments  above  mentioned  are  "  all-round  "  microtomes;  by  which 
is  meant  that  they  may  be  used  either  with  a  square-set  knife  or  an  obliquely- 
set  knife,  and  (Jung's  at  all  events)  will  cut  either  celloidin  sections  or 
frozen  preparations  (if  a  freezing  apparatus  be  added  to  them)  just  as  well 
us  paraffin  sections.  They  will  not,  according  to  my  experience,  cut  series 
of  paraffin  sections  with  anything  like  the  same  infallible  regularity, 
oertainly  not  with  the  same  rapidity  as  the  instruments  next  to  be  mentioned. 
But  they  give  excellent  results,  and  in  view  of  their  adaptability  to  celloidin 
or  other  semi-soft  preparations,  I  think  that  one  of  them,  the  Jung  by  pre- 
ference, should  be  the  instrument  chosen  by  the  worker  who  desires  not  to 
be  entirely  confined  to  the  paraffin  method,  and  who  cannot  conveniently 
possess  more  than  one  microtome. 

All  the  instruments  mentioned  hitherto  are  sliding  microtomes,  that  is 
instruments  in  which  the  object  to  be  cut  is  a  fixture  during  cutting,  and 
the  knife  is  moved  on  a  slide  and  is  only  attached  to  its  holder  at  one  end. 
This  arrangement  will  not  allow  the  highest  possible  accuracy  to  be  obtained 
"with  paraffin  objects  or  any  other  hard  objects.  For  with  hard  objects  the 
knife  is  free  to  yield  slightly  on  meeting  the  object,  instead  of  cutting  its 
way  through  it.  This  defect  is  fatal  to  the  attainment  of  perfectly  cut 
series  of  sections  of  equal  thickness  throughout.  For  the  highest  class  of 
work  it  is  necessary  to  employ  a  microtome  constructed  on  the  opposite 
principle,  namely  one  in  which  the  knife  is  a  fixture,  and  fixed  at  both  ends 
as  near  as  possible  to  the  cutting  point ;  the  object  being  moved  against  it. 
The  following  instruments  are  constructed  on  this  principle,  and  for  accurate 
cutting  of  paraffin  sections  seem  to  me  in  general  superior  to  any  sliding 
microtome.  They  also  work  incomparably  quicker;  and  for  this,  and  other 
reasons,  are  better  adapted  than '  the  sliding  microtomes  for  cutting 
continuous  ribbons  of  sections.  They  cannot  be  considered  to  be  "  all- 
round  "  instruments,  because  (although  some  of  them  are  fitted  with  an 
arrangement  for  that  purpose)  they  are  not  well  adapted  for  giving  to  the 
knife  the  oblique  position  and  slow  motion  requisite  for  cutting  objects  of  < 
very  hard,  or  very  soft,  or  very  heterogeneous  consistency.  Also,  the  object 
is  placed  in  an  awkward  position  for  orientation  and  observation  whilst 
cutting. 


IMBEDDING   METHODS.  89 

The  Cambridge  rocking  microtome  (furnished  by  the  Cambridge  Scientific 
Instrument  Company,  Carlyle  Road,  Cambridge,  price  £5,  or  by  Messrs. 
Swift  and  Son,  or  by  Jung)  is  only  adapted  for  cutting  paraffin  sections  (Mr. 
Swift  has  shown  me  an  arrangement  for  inclining  the  knife  so  as  to  give  it 
the  position  required  for  cutting  celloidin  ;  but  I  feel  pretty  sure  that  this 
will  prove  a  failure  in  practice).  This  instrument  is  extremely  simple  and 
extremely  rapid,  and,  what  is  more  important,  cuts  more  level  series  of 
sections  than  any  other  microtome  I  am  personally  acquainted  with.  It 
should  be  fitted  with  an  adjustable  object-holder,  allowing  of  precise  orienta- 
tion of  the  object.  This,  I  believe,  has  been  done  in  the  newest  (1900) 
model.  Or  the  object-holder  of  Henneguy  and  Vignal  (Compt.  Rend.  Soc. 
BioL,  1885,  p.  647)  may  be  added  to  it.  (This,  as  well  as  the  entire  instru- 
ment, is  manufactured  in  France  by  Dumaige,  9,  Rue  de  la  Bucherie,  Paris, 
or  Messrs.  Swift  on  request  will  furnish  such  an  arrangement,  or  it  may  be 
obtained,  with  or  without  the  entire  instrument,  from  Jung,  of  Heidelberg.) 
See  also  in  Zeit.  f.  wiss.  Mik.,  iv,  4,  1887,  p.  465,  the  description  of  an 
object-holder  adapted  to  the  rocking  microtome  by  HASSELAEE  ;  further,  the 
price  list  of  JUNG;  also  a  paper  in  Zeit.f.  wiss.  Mik.,  vii,  2,  1890,  p.  165. 
Jung's  form  of  the  microtome  is  more  expensive  than  the  English  one,  but 
contains  several  very  useful  improvements  in  details. 

It  has  been  objected  to  this  instrument  by  Schiefferdecker  (see  Zeit.  f. 
wiss.  Mik.,  ix,  2,  1892,  p.  171,  a  description  and  criticism  of  the  instrument 
as  made  by  Jung)  that  it  does  not  cut  plane  sections,  but  sections  having 
the  form  of  segments  of  a  cylinder.  This  is  true  ;  but  in  practice  the  slight 
deviation  of  the  sections  from  a  plane  figure  is  found  to  be  quite  in- 
appreciable, and  therefore  unimportant.  This  slight  defect  has  been  over- 
come in  the  special  model  of  1895 ;  but  this  costs  £8  10s.,  and  for 
ordinary  work,  such  as  does  not  require  very  large  sections,  may  be  dis- 
pensed with. 

Rather  more  costly  (£8  15s.)  is  the  MINOT  microtome  made  by  E. 
Zirnrnermann,  Mechaniker,  21,  Emilien  Strasse,  Leipzig.  A  description 
and  figures  of  this  instrument  will  be  found  in  Zeit.  f.  wiss.  Mik.,  ix,  2, 
1892,  p.  176,  or  in  Journ.  Roy.  Hie.  Soc.,  1889,  p.  143.  This  microtome 
cuts  plane  sections.  It  cuts  with  very  great  rapidity.  It  is  said  that 
owing  to  the  construction  of  the  slide,  which  is  subject  to  uncompensated 
wear  and  tear,  its  work  is  liable  to  fail  in  accuracy.  The  object-holder 
does  not  appear  to  be  so  scientifically  constructed  as  the  Naples  one.  Like 
the  Cambridge  instrument,  this  microtome  is  only  adapted  for  paraffin 
work. 

MAYEE  (Grundzuge)  gives  the  preference  to  the  form  of  this  instrument 
made  by  A.  Becker,  of  Gottingen,  over  that  made  by  Zimmermann. 

The  most  beautiful  of  all  these  instruments  is  the  REINHOLD-GILTAY. 
It  is  constructed  on  essentially  the  same  principle  as  the  Minot,  but  the 
detail  has  been  further  elaborated,  with  the  result  of  obtaining  an  instru- 
ment that  is  at  the  same  time  more  precise  in  operation  and  more  resistant 
to  wear  and  tear,  all  working  parts  being  compensated  throughout. 

It  has  an  arrangement  for  allowing  the  cutting  of  collodion  material.  I 
am  unable  to  say  whether  this  is  a  success.  It  is  made  by  J.  W.  GILTAY, 
Delft,  and  costs  about  £20.  A  description  will  be  found  in  Zeit.  f.  wiss. 


90  CHAPTER    VII. 

Jtfifc.,  ix,  4,  1893,  p.  445,  and  in  Journ.  Roy  Mic.  Soc.,  1893,  p.  706;  and 
a  description  of  some  later  improvements  in  Zeit.f.  wiss.  Mik.,  xv,  1,  1898, 
p.  23,  or  Journ.  Roy.  Mic.  Sue.,  1899,  p.  106. 

125.  Imbedding  Methods. — The  processes  known  as  Im- 
bedding Methods  are  employed  for  a  twofold  end.  Firstly, 
they  enable  us  to  surround  an  object,  too  small  or  too  delicate 
to  be  firmly  held  by  the  fingers  or  by  any  instrument,  with 
some  plastic  substance  that  will  support  it  on  all  sides  with 
firmness  but  without  injurious  pressure,  so  that  by  cutting 
sections  through  the  composite  body  thus  formed,  the  in- 
cluded object  may  be  cut  into  sufficiently  thin  slices  without 
distortion.  Secondly,  they  enable  us  to  fill  out  with  the  im- 
bedding mass  the  natural  cavities  of  the  object,  so  that  their 
lining  membranes  or  other  structures  contained  in  them  may 
be  duly  cut  in  situ;  and,  further,  they  enable  us  not  only  to 
surround  with  the  supporting  mass  each  individual  organ  or 
part  of  any  organ  that  may  be  present  in  the  interior  of  the 
object,  but  also  "to  fill  out  or  impregnate  with  it  each  separate 
cell  or  other  anatomical  element,  thus  giving  to  the  tissues  a 
consistency  they  could  not  otherwise  possess,  and  ensuring 
that  in  the  thin  slices  cut  from  the  mass  all  the  minutest 
details  of  structure  will  precisely  retain  their  natural  relations 
of  position. 

These  ends  are  usually  attained  in  one  of  two  ways.  Either 
•  the  object  to  be  imbedded  is  saturated  by  soaking  with  some 
material  that  is  liquid  while  warm  and  solid  when  cold, 
which  is  the  principle  of  the  processes  here  called  Fusion 
Imbedding  Methods  ;  or  the  object  is  saturated  with  some 
substance  which  whilst  in  solution  is  sufficiently  fluid  to 
penetrate  the  object  to  be  imbedded,  whilst  at  the  same 
time,  after  the  evaporation  or  removal  by  other  means  of  its 
solvent,  it  acquires  and  imparts  to  the  imbedded  object 
sufficient  firmness  for  the  purpose  of  cutting.  The  collodion 
process  sufficiently  exemplifies  this  principle.  If  a  piece  of 
soft  tissue  be  dehydrated,  and  soaked  first  in  ether  and  then 
in  collodion,  and  if  the  ether  contained  in  the  collodion  be 
allowed  slowly  to  evaporate,  the  tissue  and  mass  of  collodion 
which  penetrates  and  surrounds  it  will  acquire  a  consistency 
such  as  to  admit  of  thin  sections  being  cut  from  them.  The 
methods  founded  on  this  principle  are  here  called  Evaporation 
Imbedding  Methods. 


IMBEDDING    METHODS.  91 

In  any  of  these  cases  the  material  used  for  imbedding  is 
technically  termed  an  "imbedding  mass  " — Einbettungsmasse 
— masse  d'inclusion.  Imbedding  methods  are  spoken  of  by 
French  writers  as  methodes  d} inclusion ,  or  methodes  d'enrob- 
age. 

As  before  stated,  the  method  most  generally  employed, 
and  the  one  which  may  be  considered  the  normal  anatomical 
method,  is  the  paraffin  method. 

126.  Imbedding  Manipulations. — Imbedding  in  a  melted 
mass,  such  as  paraffin,  is  performed  in  one  of  the  following 
ways.  A  little  tray  or  box  or  thimble  is  made  out  of  paper, 
some  melted  mass  is  poured  into  it ;  at  the  moment  when 
the  mass  has  cooled  so  far  as  to  have  a  consistency  that  will 
not  allow  the  object  to  sink  to  the  bottom,  the  object  is 
placed  on  its  surface,  and  more  melted  mass  poured  on  until 
the  object  is  covered  by  it.  Or  the  paper  tray  being  placed 
on  cork,  the  object  may  be  fixed  in  position  in  it  whilst 
empty  by  means  of  pins  and  the  tray  filled  with  melted 
mass  at  one  pour.  The  pins  are  removed  when  the  mass  is 
cold. 

In  either  case,  when  the  mass  is  cold  the  paper  is  removed 
from  it  before  cutting. 

To  make  paper  trays,  proceed  as  follows.  Take  a  piece 
of  stout  paper  or  thih  cardboard,  of  the  shape  of  the 
annexed  figure  (Fig.  1)  ;  thin  (foreign)  post- cards  do  very 
well  indeed.  Fold  it  along  the  lines  a  a  and  b  b',  then  along 
c  c  and  d  d',  taking  care  to  fold  always  the  same  way. 
Then  make  the  folds  A  A',  B  B' ,  C  C',  D  D' ',  still  folding  the 
same  way.  To  do  this  you  apply  A  c  against  A  a,  and 
pinch  out  the  line  A  A',  and  so  on  for  the  remaining  angles. 
This  done,  you  have  an  imperfect  tray  with  clogs'  ears  at 
the  angles.  To  finish  it,  turn  the  dogs'  ears  round  against 
the  ends  of  the  box,  turn  down  outside  the  projecting  flaps 
that  remain,  and  pinch  them  down.  A  well-made  post-card 
tr;i y  will  last  through  several  imbeddings,  and  will  generally 
work  better  after  having  been  used  than  when  new. 

Another  method  of  folding  the  paper  (MAYER)  is  described 
in  the  Grundziige,  p.  72. 

To  make  paper  thimbles,  take  a  good  cork,  twist  a  strip  of 
paper  several  times  round  it  so  as  to  make  a  projecting 


•92 


CHAPTER    VII 


Collar,  and  stick  a  pin  through  the  bottom  of  the  paper  into 
the    cork.      For  work  with  fluid  masses,  such  as   celloidin, 


A' 


~ 


\ 


A 


._£ 

B| 


FIG.  2. 


FIG.  1. 


the  cork  may  be  loaded  at  the  bottom  by  means  of  a  nail 
or  piece  of  lead,,  to  prevent  it  from  floating  when  the  whole 
is  thrown  into  spirit  or  other  liquor  for  hardening  (Fig.  2). 
LEUCKHART'S  Imbedding  Boxes  are  made  of  two  pieces  of 
type-metal  (Fig.  3).  Each  of  these  pieces  has  the  form  of 
a  carpenter's  "  square "  with  the  end  of  the  shorter  arm 
triangularly  enlarged  outwards.  The  box  is  constructed  by 
placing  the  two  pieces  together  on  a  plate  of  glass  which 
has  been  wetted  with  glycerin  and  gently  warmed.  The 
area  of  the  box  will  evidently  vary  according  to  the  position 
given  to  the  pieces,  but  the  height  can  be  varied  only  by 
using  different  sets  of  pieces.  Two  sets  will  be  sufficient 
for  most  work  ;  one  set  of  one  centimetre  in  height,  and  one 
of  two  centimetres,  each  being  eight  centimetres  in  length, 
and  three  in  breadth.  To  make  the  box  paraffin -tight,  so 
that  it  will  hold  the  melted  paraffin  long  enough  in  the 
liquid  state  to  permit  of  the  objects  being  carefully  orientated 
in  it,  MAYER  (Mitth.  Zool.  Stat.  Neapel,iv,  1883,  p.  429)  first 


IMBEDDING    METHODS. 


93 


FIG.  3. 


smears   the   glass   plate   with   glycerin,   then   arranges 

metal  "  squares,"  and  then  fills  the  box  with  collodion,  which 

is  poured  out  again  immediately.       As 

the   ether   evaporates,    a  thin  layer  of 

collodion  remains  behind,  which  suffices 

to  keep   the  paraffin  from  running  out. 

Even   without  the    collodion,  the  mere 

cooling    of  the   paraffin   by    the   metal 

will  generally  suffice  to  keep  it  in  long 

enough  for  orientation,  if  it  is  not  in  a 

superheated  state  when  it  is  poured  in. 

In  such  a  collodionised  box  the 
paraffin  may  be  kept  in  a  liquid  state 
by  warming  now  and  then  over  a  spirit 
lamp,  and  small  objects  be  placed  in 
any  desired  position  under  the  micro- 
scope (Journ.  Roy.  Mic.  Soc.  [N.S.], 
ii,  p.  880). 

A  lighter  form  of  "  squares/'  made  of  brass,  and  devised 
by  ANDRES,  G-IESBRECHT,  and  MAYEE,  is  described  loc.  cit.  (see 
Journ.  Roy.  Mic.  Soc.,  1883,  p.  913). 

FRANKL  (Zeit.  f.  wiss.  Mile.,  xiii,  1897,  p.  438)  builds  up 
boxes  with  rectangular  blocks  of  glass,  which  may  be  found 
convenient,  but  are  more  expensive  than  the  metal  squares. 

SELENKA  has  described  and  figured  another  sort  of  appa- 
ratus having  the  same  object.  It  consists  of  a  glass  tube, 
through  which  a  stream  of  warm  water  may  be  passed  and 
changed  for  cold  as  desired,  the  object  being  placed  in  a 
depression  in  the  middle  of  the  tube  (see  Zool.  Anz.,  1885, 
p.  419).  A  simple  modification  of  this  apparatus,  which 
anyone  may  make  for  himself,  is  described  by  ANDREWS  in 
Amer.  Natural.,  1887,  p.  101  ;  cf.  Zeit.  f.  wiss.  Mik.,  iv,  3, 
1887,  p.  375;  or  Journ.  Roy.  Mic.  Soc.,  1887,  p.  510;  and  a 
more  complicated  imbedding  and  orienting  box,  seldom 
necessary,  is  described  by  JORDAN,  in  Zeit.  f.  wiss.  Mik.,  xvi, 
1,  1899,  p.  32 ;  Journ.  Roy.  Mic.  Soc.,  132,  1899,  p.  549. 

For  small  paraffin  objects  the  following  procedure  is  very 
useful.  The  object  is  removed  from  the  melted  paraffin, 
the  superfluous  paraffin  is  removed  by  means  of  blotting- 
paper,  and  the  object  placed  on  a  cylinder  of  paraffin.  A 
piece  of  stout  iron  wire  is  now  heated  in  the  flame  of  a 


94  CHAPTER   VII. 

spirit  lamp,  and  with  it  a  hole  is  melted  in  the  end  of  the 
cylinder;  the  specimen  is  pushed  into  the  melted  paraffin, 
and  placed  in  any  desired  position.  The  advantages  of  the 
method  lie  in  the  quickness  and  certainty  with  which  it  can 
be  performed. 

There  remains  the  watch  glass  method,  Melt  paraffin  in  a 
watch  glass,  and  throw  the  object,  previously  well  de- 
alcoholised  and  penetrated  with  a  solvent,  into  it ;  or  place 
the  object  in  the  watch  glass,  add  solid  paraffin,  and  heat. 
After  the  mass  has  hardened,  cut  out  a  block  containing  the 
object  (this  is  of  course  applicable  to  other  masses,  such  as 
celloidin).  This  should  be  done  with  a  thin-bladed  knife, 
slightly  warmed.  If  paraffin  be  used  you  may,  instead  of 
cutting  out  a  block,  turn  out  the  whole  mass  of  paraffin  by 
simply  warming  rapidly  the  bottom  of  the  glass,  but  I  find 
it  is  far  safer  to  cut  out  a  block.  To  facilitate  the  removal 
of  the  mass  some  persons  lubricate  the  watch  glass  before 
pouring  in  the  mass.  To  do  this  a  drop  of  glycerin  or, 
according  to  some,  clove  oil,  should  be  smeared  over  it  and 
wiped  off  with  a  cloth  until  hardly  a  trace  of  it  remains. 
But  this  is  not  necessary. 

As  regards  small  objects  at  all  events,  I  consider  the 
watch  glass  process  to  be  the  very  best  of  any. 

For  imbedding  very  small  objects  in  this  way  certain  precautions  may  be 
necessary  in  order  not  to  lose  them.  SAMTEE  (Zeit.f.  iviss.  Mik.,  xi,  1894, 
p.  469)  saturates  small  unstained  objects  with  paraffin  that  has  previously 
been  strongly  coloured  with  alkanna  extract,  and  then  imbeds  them  in  pure 
paraffin.  The  objects  do  not  stain  with  the  alkanna.  KHUMBLEE  (ibid., 
xii,  1895,  p.  312 ;  and  xiii,  3,  1896,  p.  303)  stains  previously  the  objects 
themselves  lightly  with  eosin  dissolved  in  strong  alcohol,  and  removes  the 
stain  from  the  sections  with  weak  alcohol.  See  also  ibid.,  xiii,  2,  p.  200,  a 
paper  by  SCHYDLOWSKI  ;  and  in  Zeit.  f.  wiss.  Zool.,  Iviii,  1897,  p.  144,  a 
process  of  BOEGEBT. 

127.  Choice  of  a  Method. — Amongst  the  very  various 
methods  of  imbedding  that  have  been  proposed  two  are  pre- 
eminently important — the  paraffin  method  for  small  objects, 
and  the  celloidin  or  collodion  method  for  large  objects ; 
indeed  these  are  the  only  ones  that  have  survived  in  general 
use. 

The  subject  of  the  respective  merits  of  paraffin  and  cel- 
olidin  still  affords  matter  for  discussion.  The  case,  however, 


IMBEDDING   METHODS.  95 

seems  to  be  a  very  simple  one.  Celloidin  does  not  afford  by 
a  long  way  the  thinnest  sections  that  are  obtainable  with 
small  objects.  For  such  objects  it  is,  therefore,  not  equal  to 
the  demands  made  by  modern  minute  anatomy,  and  paraffin 
must  be  taken.  On  the  other  hand,  paraffin  (as  at  present 
employed)  will  only  cut  very  thin  sections  with  fairly  small 
objects ;  with  objects  of  much  over  half  an  inch  in  diameter 
you  cannot  get  with  paraffin  thinner  sections  than  you  can 
with  celloidin ;  and  if  you  try  to  cut  in  paraffin  objects  of 
still  greater  size,  say  an  inch  and  upwards,  it  will  frequently 
happen  that  you  will  not  get  perfect  sections  at  all,  blocks 
of  paraffin  of  this  size  having  a  tendency  to  split  under  the 
impact  of  the  knife ;  so  that  for  very  large  objects  celloidin 
generally  gives  better  results,  in  this  respect,  besides  pre- 
senting certain  advantages  for  the  manipulation  and  staining 
of  the  sections. 

This  defect  is,  however,  much  reduced  by  the  employ- 
ment of  a  softer  paraffin  than  is  usual.  In  this  way  STEASSER 
(Zeit.  f.  iviss.  Mik.,  ix,  1892,  p.  7)  has  obtained  series  of 
frontal  sections  30  /u  thick  through  the  entire  human  brain, 
in  paraffin  blocks  measuring  10  x  15  cm. 

I  have  not  been  able  to  satisfy  myself  that  the  preserva- 
tion of  the  tissues  is  better  in  celloidin  sections  than  in 
paraffin  sections  ;  so  that — convenience  apart — the  case 
remains  as  above  stated, — paraffin  for  small  sections,  cel- 
loidin for  large  ones. 

To  these  may  be  added  aqueous  masses,  such  as  gum  or 
gelatin,  for  very  special  cases. 


CHAPTER  VIII. 

IMBEDDING  METHODS— PARAFFIN  AND  OTHER  FUSION  MASSES. 

128.  Saturation  with  a  Solvent. — The  first  stage  of  the 
paraffin  method  consists  in  the  saturation  of  the  object  with 
some  substance  which  is  a  solvent  of  paraffin.  The  process 
is  sometimes  called  a  clearing  process,  since  many  of  the 
substances  used  for  infiltration  are  also  "  clearing  "  agents. 

The  process  of  saturation  should  be  carefully  performed 
with  well-dehydrated  objects  in  the  manner  described  in 
§  106. 

Saturation  liquids  being  liquids  that  are,  on  the  one  hand, 
miscible  with  alcohol,  and  on  the  other  hand  good  solvents 
of  paraffin,  are  not  quite  as  numerous  as  could  be  wished. 
Amongst  them  may  be  mentioned  essence  of  turpentine,  clove 
oil,  bergamot  oil,  benzol,  xylol,  toluol,  naphtha,  oil  of  cedar- 
wood,  chloroform,  and  anilin  oil.  But  they  are  by  no  means 
all  equally  good,  for  few  of  them  are  as  good  solvents  of 
paraffin  as  is  desirable. 

Turpentine  penetrates  well,  and  mixes  readily  with  paraffin. 
I  do  not,  however,  recommend  it,  because  in  my  experience 
it  is  of  all  others  the  clearing  agent  that  is  the  most  hurtful 
to  delicate  structures. 

Clove  oil  penetrates  well,  and  preserves  delicate  structures 
well ;  but  it  mixes  very  imperfectly  with  paraffin,  and  quickly 
renders  tissues  brittle. 

Benzol  has  been  recommended  by  BRASS  (Zeit.f.  wiss.  Mi'k., 
ii,  1885,  p.  301). 

Toluol  (or  toluen)  has  been  recommended  by  HOLL  (Zool. 
Anz.,  1885,  p.  223). 

Naphtha  has  been  recommended  by  WEBSTEE  (Journ.  Anat.  and  Physiol., 
xxv,  1891,  p.  278).     For  large  specimens  it  has  the  advantage  of  being  very 


IMBEDDING   METHODS    (PARAFFIN).  97 

cheap.  Dr.  Webster  writes  me  that  a  quality  known  as  "Persian  naphtha  " 
is  best  for  fine  work,  but  the  common  pure  naphtha  is  sufficient  for  ordinary 
work. 

FIELD  and  MARTIN  (Zeit.f.  wiss.  Mik.,  xi,  1,  1894,  p.  10)  recommend  a 
light  petroleum  known  as  "  petroleum-aether." 

Xylol  is  said  by  M.  HEIDENHAIN  (Kern  und  Protoplasma, 
p.  114)  to  be  a  cause  of  shrinkage  in  cells ;  he  employs  oil  of 
bergamot.  This,  according  to  APATHY  (Mihrotechnik,p.  117), 
only  dissolves  very  little  paraffin. 

For  Gueguen's  methyl  salicylate,  see  §  123a. 

Chloroform  mixes  well  with  paraffin,  and  after  evaporation 
in  a  paraffin  bath  (in  the  manner  described  in  the  next  para- 
graph) leaves  behind  a  pure  and  very  homogeneous  paraffin, 
having  but  little  tendency  to  crystallise.  But  it  is  deficient 
in  penetrating  power,  so  that  it  requires  an  excessive  length 
of  time  for  clearing  objects  of  any  size ;  and  it  must  be  very 
thoroughly  got  rid  of  by  evaporation  in  the  paraffin  bath,  or 
by  successive  baths  of  paraffin,  as  if  the  least  trace  of  it 
remains  in  the  paraffin  used  for  cutting  it  will  make  it  soft. 
The  process  of  removal  requires  a  very  long  time,  in  some 
cases  days.  Chloroform  ought  therefore  to  be  reserved  for 
small  and  easily  penetrable  objects.  Under  suitable  con- 
ditions, and  properly  employed,  it  is  certainly  one  of  the  best, 
if  not  the  very  best  of  these  media. 

Cedar-wood  oil  is,  according  to  my  continued  experience,' 
for  the  reasous  stated  by  me  in  Zool.  Anz.,  1885,  p.  563,  for 
general  work  the  very  best  clearing  agent  for  paraffin  im- 
bedding. It  penetrates  extremely  rapidly,  preserves  delicate 
structure  better  than  any  clearing  agent  known  to  me,  does 
not  make  tissues  brittle,  even  though  they  may  be  kept  for 
weeks  or  months  in  it,  and  has  the  great  advantage  that  if  it 
be  not  entirely  removed  from  the  tissues  in  the  paraffin  bath 
it  will  not  seriously  impair  the  cutting  consistency  of  the 
mass ;  indeed,  I  fancy  it  sometimes  improves  it  by  rendering 
it  less  brittle.  I  do  not  mean  to  assert  that  it  is  in  all  cases 
the  best,  for  for  some  fine  work  I  think  chloroform  may  give 
more  accurate  results.  And  it  may  often  be  indicated  to 
employ  the  two  reagents  successively,  as  recommended  by 
APATHY,  see  next  §. 

In     some     difficult     cases    anilin    oil    is     indicated    (see 


98  CHAPTER    VIII. 

129.  The  Paraffin  Bath. — The  objects  having  been  duly 
saturated  with  a  solvent,  the  next  step  is  to  substitute 
melted  paraffin  for  the  saturating  medium. 

Some  authors  lay  great  stress  on  the  necessity  of  making 
the  passage  from  the  saturating  agent  to  the  paraffin  as 
gradual  as  possible,  by  means  of  successive  baths  of  mixtures 
of  solvent  and  paraffin  kept  melted  at  a  low  temperature, 
say  35°  C.  With  oil  of  cedar  or  toluol,  at  all  events,  this  is 
not  necessary.  All  that  is  necessary  is  to  bring  the  objects 
into  melted  paraffin  kept  just  at  its  melting-point,  and  keep 
them  there  till  they  are  thoroughly  saturated;  the  paraffin 
being  changed  once  or  twice  for  fresh  only  if  the  objects  are 
sufficiently  voluminous  to  have  brought  over  with  them  a 
notable  quantity  of  clearing  agent. 

The  practice  of  giving  successive  baths  first  of  soft  and  then 
of  hard  paraffin  appears  to  me  entirely  illusory. 

It  is  important  to  keep  the  paraffin  dry — that  is  protected 
from  vapour  of  water  during  the  bath. 

It  is  still  more  important  to  keep  it  as  nearly  as  possible 
at  melting-point.  If  it  be  heated  for  some  time  to  a  point 
much  over  its  normal  melting-point,  the  melting-point  will 
rise,  and  you  will  end  by  having  a  harder  paraffin  than  you 
set  out  with.  And  as  regards  the  preservation  of  tissues,  of 
course  the  less  they  are  heated  the  better.  Overheating,  as 
well  as  prolonged  heating,  tends,  amongst  other  things,  to 
make  tissues  brittle. 

The  duration  of  the  bath  must,  of  course,  vary  according 
to  the  size  and  nature  of  the  object.  An  embryo  of  2  to  3 
millimetres  in  thickness  ought  to  be  thoroughly  saturated 
after  an  hour's  bath,  or  often  less,  if  cedar  oil  has  been  used 
for  clearing.  Many  workers  habitually  give  much  longer 
baths,  I  think  often  longer  than  necessary.  I  take  as  a 
guide,  generally,  the  length  of  time  the  object  has  taken  to 
clear  iu  the  cedar  oil,  assuming  that  the  inarm  melted  paraffin 
r  ought  to  penetrate  at  least  as  quickly  as  the  cold  oil  ;  and 
then  allowing  somewhat  longer,  say  as  much  again,  in  order 
to  be  on  the  right  side. 

In  any  case  the  preparations  should  be  cooled  (see  below, 
§  132)  as  soon  as  saturated.  If  left  for  very  many  hours  in 
a  warm  bath,  as  is  sometimes  done,  delicate  structures  may 
be  seriously  injured.  ,Who  can  say  what  is  not  dissolved 


IMBEDDING    MKTHODS     (  PARAFFIN).  99 

out  of  cells  by  prolonged  heating  in  mixtures  of  paraffin 
with  chloroform  or  benzol,  or  the  like  ?  Indeed,  it  seems 
to  me  that  the  great  point  to  be  attended  to  in  paraffin  work 
of  the  finer  order  is  to  minimise  the  action  of  heat.  It  is 
therefore  important  both  to  employ  a  paraffin  of  the  lowest 
melting-point  that  will  give  good  sections  (see  below,  §  141), 
and  to  abbreviate  the  warm  bath  as  much  as  possible. 

If  chloroform  or  other  volatile  agent  be  taken,  choice  may 
be  rnao^eoftwo  methods  :  either,  as  in  Giesbrecht's  method, 
the  chloroform  containing  the  object  is  heated  to  the  melting- 
point  of  the  paraffin,  and  the  paraffin  gradually  added,  and 
the  mass  kept  at  the  melting-point  of  the  pure  paraffin  until 
all  the  chloroform  is  driven  off ;  or,  as  in  Biitschli's  method, 
the  objects  are  simply  passed  direct  from  chloroform  into  a 
solution  of  paraffin  in  chloroform,  in  which  they  remain  until 
thoroughly  impregnated  (half  to  one  hour),  and  which  is  then 
evaporated  at  the  melting-point  of  the  paraffin.  Biitschli 
recommends  a  paraffin  solution  melting  at  35°.  (Such  a 
solution  is  made  of  about  equal  parts  of  chloroform  and 
paraffin  of  50°  melting-point.)  Or,  in  the  case  of  larger 
objects,  instead  of  evaporating  the  chloroform  (which  is  often 
a  very  long  process,  as  the  chloroform  must  be  completely 
driven  off,  or  the  mass  will  remain  too  soft  for  cutting), 
Biitschli  simply  transfers  them  from  the  bath  of  paraffin  ! 
solution  to  a  bath  of  pure  paraffin. 

Giesbrecht's  method  (Zool.  AHZ.,  1881,  p.  484),  more  fully 
stated,  is  as  follows  : 

Objects  to  be  imbedded  are  saturated  with  absolute  alcohol 
and  then  brought  into  chloroform  (to  which  a  little  sulphuric 
ether  has  been  added  if  necessary,  in  order  to  prevent  the 
objects  from  floating).  As  soon  as  the  objects  are  saturated 
wi  ch  the  chloroform,  the  chloroform  and  objects  are  gradually 
warmed  up  to  the  melting-point  of  the  paraffin  employed,  and 
during  the  warming  small  pieces  of  paraffin  are  by  degrees 
added  to  the  chloroform.  So  soon  as  it  is  seen  that  no  more 
bubbles  are  given  off  from  the  objects,  the  addition  of  paraffin 
mjiv  cease,  for  that  is  a  sign  that  the  paraffin  has  entirely 
displaced  the  chloroform  in  the  objects.  This  displacement 
having  been  a  gradual  one,  the  risk  of  shrinkage  of  the 
tissues  is  reduced  to  a  minimum. 

MAYER  (Grundzuge,  p.  78)  first  saturates  the  objects  with 


100  CHAPTER   VIII. 

benzol,  which  should  be  changed  once  or  twice  so  as  to  make 
sure  of  removing  all  the  alcohol,  and  then  adds  to  the  pure 
benzol  some  small  pieces  of  paraffin,  and  lets  them  dissolve 
in  the  cold.  After  several  hours  (up  to  eighteen)  the  whole 
is  brought  in  an  open  vessel  on  to  the  cold  water-bath,  the 
bath  is  then  warmed  gradually  so  as  to  attain  a  temperature 
of  60°  C.  in  about  two  hours,  and  as  fast  as  the  benzol 
evaporates  melted  paraffin  is  added  to  it.  Lastly,  the  paraffin 
is  changed  once  before  the  definitive  imbedding.  He  rarely 
leaves  objects  over  night  in  the  water-bath. 

APATHY  (Mikrotechnik,  pp.  149-150)  first  clears  with  oil 
of  cedar,  then  brings  the  objects  (by  the  process  described 
§  106)  into  a  solution  of  paraffin  in  chloroform  saturated  at 
the  temperature  of  the  laboratory.  The  objects  remain  in 
the  chloroform-paraffin  solution  for  from  one  to  three  hours, 
without  warming,  until  all  the  cedar  oil  is  soaked  out  of 
them.  The  whole  is  then  warmed  on  the  water-bath  or  oven 
to  a  few  degrees  above  the  melting-point  of  the  paraffin 
intended  to  be  used  for  imbedding,  and  the  object  is  brought 
into  a  mixture  of  equal  parts  of  paraffin  and  chloroform, 
being  suspended  therein  near  the  top  on  a  bridge  made  of 
hardened  filter  paper  (or  in  a  special  apparatus  to  the  same 
end,  not  yet  described).  It  remains  in  this  mixture,  at  the 
temperature  of  the  oven,  for  one  to  three  hours,  and  lastly 
is  brought  (still  on  the  paper  bridge  or  in  the  apparatus) 
into  pure  paraffin,  where  it  remains  for  half  an  hour  to  two 
hours. 

HEIDENHATN  (Festschr.  f.  Koelliker,  Leipzig,  1892,  p.  114) 
clears  with  bergamot  oil,  and  passes  into  pure  paraffin 
through  mixtures  of  the  oil  and  paraffin.  RABL  also  (Zeit. 
f.  wiss.  Mik.,  xi,  1894,  p.  164)  employs  bergamot  oil. 

\v 
130.  Water-baths   and   Ovens. — It   is   important  that  the   paraJfin 

should  not  be  exposed  to  a  moist  atmosphere  whilst  it  is  in  the  liquid  state. 
If  a  water-bath  be  used  for  keeping  it  at  the  required  temperature,  provision 
should  be  made  for  protecting  the  paraffin  from  the  steam  of  the  heated 
water. 

A  very  convenient  apparatus  for  this  purpose  is  that  of  Paul  Mayer,  or 
"  Naples  water-bath,"  which  will  be  found  described  at  p.  146  of  Journ.  Roy. 
Mic.  Soc.,  1883.  It  may  be  procured  from  the  opticians,  e.  g.  Mr.  Baker. 
See  also  Amer.  Natural.,  1886,  p.  910  ;  and  Journ.  Roy.  Mic.  Soc.,  1887, 
p.  167. 


IMBEDDING    METHODS    (PARAFFIN).  101 

Other  similar  forms  of  paraffin  heating  apparatus  are  described  in  several 
places  in  the  same  journal,  as  also  in  Zeit.  f.  iviss.  Mile. 

Amongst  apparatus  arranged  for  heating  by  means  of  petroleum  or  similar 
combustibles  other  than  gas  may  be  mentioned  the  stove  manufactured  and 
sold  by  F.  SANTOBIUS,  Gottingen  (Zeit.  f.  wiss.  Mik.,  x,  2,  1893,  p.  161), 
and  that  of  ALTMANN  (ibid.,  p.  221,  cf.  Centralb.  f.  BaUeriol.,  xii,  1892, 
p.  654);  also  that  of  KABAWAIEW,  Zeit.f.  wiss.  Mik.,  xiii,  2,  1896,  p.  172. 

For  ordinary  work  it  is  by  no  means  necessary,  though  it  may  be  con- 
venient, to  possess  one  of  these  costly  and  complicated  heat-regulating  con- 
trivances ;  a  spirit  lamp  with  the  wick  well  turned  down,  or  a  night-light, 
will  suffice  to  keep  the  temperature  constant  enough,  if  watched  occasionally. 

Ovens  are,  I  think,  only  necessary  in  laboratories  where  several  students 
have  to  work  with  the  same  apparatus ;  for  the  private  worker  I  think  the 
water-bath  is  more  convenient. 

131.  Imbedding  ix  VACUO. — There  are  objects  which,  on  account  of  their 
consistency  or  their  size,  cannot  be  penetrated  by  paraffin  in  the  ordinary 
way,  even  after  hours  or  days  in  the  bath.  For  such  objects  the  method  of 
imbedding  under  a  vacuum  (strictly,  under  diminished  atmospheric  pressure), 
renders  the  greatest  service.  It  not  only  ensures  complete  penetration  in  a 
very  short  time— a  few  minutes— but  it  has  the  further  advantage  of  pre- 
venting any  falling  in  of  the  tissues,  such  as  may  easily  happen  with  objects 
possessing  internal  cavities  if  it  be  attempted  to  imbed  them  in  the  ordinary 
way. 

The  principle  of  this  method  is  that  the  objects  are  put  through  the 
paraffin  bath  in  vacua.  In  practice  this  may  be  realised  by  means  of  any 
arrangement  that  will  allow  of  maintaining  paraffin  at  the  necessary  tempe- 
rature for  keeping  it  fluid  under  a  vacuum. 

The  apparatus  of  HOFFMANN  is  described  and  figured  at  p.  230  of  Zool. 
Anz.,  1884.  In  this  arrangement  the  vacuum  is  produced  by  means  of  a 
pneumatic  water  aspiration  pump,  the  vessel  containing  the  paraffin  being 
placed  in  a  desiccator  heated  by  a  water-bath  and  furnished  with  a  tube  that 
brings  it  into  communication  with  the  suction  apparatus.  This  arrangement 
is  very  efficacious  and  very  simple  if  the  laboratory  possesses  a  supply  of 
water  under  sufficient  pressure. 

In  order  to  obtain  the  requisite  vacuum  without  the  aid  of  water  under 
pressure,  a  simple  little  apparatus  has  been  designed  by  FBANCOTTE  (Butt. 
Soc.  Belg.  Mic.,  1884,  p.  45).  In  this  the  vacuum  is  produced  by  the  con- 
densation of  steam. 

FOL  (Lehrb.,  p.  121)  employs  the  vacuum  apparatus  of  Hoffmann,  but 
simplifies  the  arrangement  for  containing  the  paraffin.  The  paraffin  is  con- 
tained in  a  stout  test-tube  furnished  with  a  rubber  stopper  traversed  by  a 
tube  that  puts  it  into  communication  with  the  pump.  The  lower  end  of  the 
test-tube  dips  into  a  water-bath.  You  pump  out  the  air  once  or  twice,  wait 
a  few  minutes  to  make  sure  that  no  more  bubbles  rise,  then  let  the  air  in, 
turn  out  the  object  with  the  paraffin  (which  by  this  time  will  have  become 
abnormally  hard),  and  re-imbed  in  fresh  paraffin. 

See  also  a  paper  by  PBIXGLE,  in  Journ.  Path,  and  Bacterial.,  1892,  p.  117  ; 
or  Journ.  Roy.  Mic.  Soc.,  1892,  p.  893. 


302  CHAPTER    VIH. 

132.  Imbedding  and  Cooling, — As  soon  as  the  objects  are 
thoroughly  saturated  with  paraffin  they  should  be  imbedded 
by  one  of  the  methods  given  above  (§  126).  If  the  watch 
glass  method  be  followed  the  paraffin  bath  will  naturally  have 
been  given  in  the  watch  glass  used  for  imbedding,  and  no 
special  imbedding  manipulation  will  be  necessary.  In  any 
case  the  important  point  now  to  be  attended  to  is  that  the 
paraffin  be  cooled  as  rapidly  as  possible.  The  object  of  this  is 
to  prevent  crystallisation  of  the  paraffin,  which  may  happen 
if  it  be  allowed  to  cool  slowly,  and  to  get  as  homogeneous  a 
mass  as  possible. 

Yery  small  objects  may  be  taken  out  of  the  paraffin  with 
a  needle  or  small  spatula,  and  put  to  cool  on  a  block  of  glass, 
then  imbedded  in  position  for  cutting  on  a  cone  of  paraffin 
by  means  of  a  heated  needle  in  the  manner  described  above 
(§  126).  In  the  use  of  the  needle  it  should  be  noted  that  it 
is  important  to  melt  as  little  paraffin  as  possible  at  one  time, 
in  order  that  that  Which  is  melted  may  cool  again  as  rapidly 
as  possible. 

For  BOVEEI'S  plan  for  imbedding  numerous  very  small  objects  see  "  Em- 
bryological  Methods,"  and  for  that  of  LATJTEBBORN  see  '"  Protozoa." 

If  the  watch  glass  method  be  adopted,  float  the  watch 
glass  with  the  paraffin  and  objects  on  to  cold  water.  Do 
not  let  it  sink  till  all  the  paraffin  has  solidified.  When  cool, 
cut  out  blocks  containing  the  objects  ;  do  this  with  a  slightly 
warmed  scalpel. 

If  paper  trays  be  taken,  cool  them  OQ  water,  holding  them 
above  the  surface  with  only  the  bottom  immersed  until  all 
the  paraffin  has  solidified,  as  if  you  let  them  go  to  the  bottom 
at  once  you  will  probably  get  cavities  filled  with  water  formed 
in  your  paraffin.  Or  you  may  put  them  to  cool  on  a  block 
of  cold  metal  or  stone. 

Preparations  imbedded  in  the  metal  "  squares  "  are  cooled 
in  a  similar  manner. 

SELENKA  cools  the  mass  by  passing  a  stream  of  cold  water  through  the 
imbedding  tube  described  above  (§  126).  MAYER  cools  the  mass  in  the 
paraffin-tight  moulds  §  126  by  passing  cold  water  through  a  special  movable 
water-bath,  which  allows  of  the  arrangement  of  the  objects  by  transmitted 
light  under  a  dissecting  microscope,  see  Mitth.  Zool.  Stat.  Neapel,  iv,  1883, 
p.  429;  Intern.  Munaisscltr.  Anat.  Hist.,  iv,  1887,  p.  39. 


IMBEDDING    MKTflODS    (PARAFFIN).  103 

The  objects  having  been  mounted  on  the  carrier  of  the 
microtome  in  position  for  cutting,  pare  the  blocks  to  the 
proper  shape  (see  below,  §  "  Cutting  ")  and  sufficiently  close 
down  to  the  objects,  and  go  round  them  with  a  lens.  If  any 
bubbles  or  cavities  or  opaque  spots  be  present,  prick  with  a 
heated  needle  till  all  is  smooth  and  homogeneous.  Minutes 
spent  in  this  way  are  well  invested. 

It  is  said  by  some  workers  that  it  is  well  to  cut  within  a 
few  hours  of  imbedding  if  the  structure  be  at  all  delicate,  as 
paraffin  may  continue  to  crystallise  slowly  to  a  certain  extent 
even  after  rapid  cooling.  But  this  danger  is  very  greatly 
diminished  if  the  mass  have  been  properly  cooled.  And 
according  to  my  experience  the  damage  likely  to  arise  from 
the  crystallisation  of  the  paraffin  has  been  greatly  exaggerated. 
As  stated  in  §  3,  I  find  no  better  medium  for  the  preserva- 
tion of  tissues  than  paraffin. 


133.  Orientation  of  the  Object. —  The  above-described  manipulations 
of  definitive  imbedding  are  in  most  cases  sufficient.  But  it  may  be  desir- 
able to  have  the  object  fixed  in  the  cooled  block  in  a  more  precisely- 
arranged  position,  and,  above  all,  in  a  more  precisely-marked  position,  than 
is  possible  by  these  simple  methods.  Here  is  a  method  due  to  PATTEN 
(Zeit.f.  wiss.  Mik.,  xi,  1,  1894,  p.  13),  which  is  especially  useful  when  one 
desires  to  orient  accurately  large  numbers  of  small  and  similar  objects. 
You  get  some  writing  paper  of  the  soil  that  is  made  with  two  sets  of  raised 
parallel  lines  running  at  right  angles  to  each  other  (according  to  WOOD- 
WOBTH.  see  below,  this  is  known  as  "  linen  cloth  paper  ").  Small  strips 
are  cut  from  this,  and  at  suitable  intervals  along  them  small  drops  of  a 
mixture  of  collodion  and  clove  oil,  of  about  the  consistency  of  thick  honey, 
are  arranged  close  together  along  one  of  the  ribs  that  run  lengthwise.  The 
objects  to  be  imbedded  are  cleared  in  clove  oil  or  oil  of  bergamot — not 
turpentine.  They  are  taken  one  by  one  on  the  point  of  a  knife,  and  after 
the  excess  of  oil  has  been  drawn  off  are  transferred  each  to  a  drop  of  the 
collodion  mixture.  They  may  be  adjusted  therein  under  the  dissecting 
microscope,  and  will  stay  in  any  required  position.  When  half  a  dozen  or 
more  objects  have  been  oriented  in  reference  to  the  cross  lines  (which  are 
to  be  parallel  to  the  section  planes)  the  whole  thing  is  placed  in  turpentine. 
This  washes  out  the  clove  oil  and  fixes  the  objects  very  firmly  to  the  paper. 
The  paper  with  the  attached  objects  is  now  passed  through  the  bath  of 
paraffin  and  imbedded  in  the  usual  way.  After  cooling  on  water  the  block 
is  trimmed  and  the  paper  peeled  off,  leaving  the  objects  in  the  paraffin 
close  to  the  under  surface  of  the  block.  This  surface  is  now  seen  to  be 
marked  by  the  orienting  lines  of  the  ribbed  paper,  and  also  by  any  record 
numbers  which  may  before  imbedding  have  been  written  with  a  soft  pencil 
on  the  paper. 


104  CHAPTER    VIII. 

A  somewhat  move  complicated  form  of  this  process  has  been  described  by 
WOODWORTH,  Bull.  Mus.  Comp.  ZooL,  xxxviii,  vol.  xxv,  1893,  p.  45. 

A  similar  process  has  also  been  described  by  FIELD  and  MARTIN  in  Zeit. 
f.  wiss.  MiJc.,  xi,  i,  1894,  p.  11,  small  strips  of  gelatin  being  used  instead  of 
paper. 

HOFFMANN  (Zeit.  f.  wiss.  Mik.,  xv,  3,  1899,  p.  312)  prefers  to  take, 
instead  of  the  ribbed  paper,  glass  slides  ruled  with  a  diamond,  and  to  com- 
pletely imbed  the  objects  in  large  drops  of  clove  oil  collodion  (equal  parts, 
allowed  to  stand  for  twenty-four  hours  in  an  open  vessel).  The  drops  are 
caused  to  set  in  xylol  instead  of  turpentine.  See  also  SAMTER,  ibid.,  xiii, 
4,  1897,  p.  441,  and  PETER,  Verh.  Anat.  Ges.,  xiii  Yers.,  1899,  p.  134. 
This  subject  is  further  treated  under  the  head  of  "  Reconstruction  from 
Sections  "  in  the  chapter  on  Embryological  Methods,  which  see. 

134.  Knife-position  and  Shape  of  Mass  to  be  Cut. — Even 
with  the  most  perfectly  imbedded  objects  it  is  frequently 
impossible  to  obtain  sections  that  are  good  in  every  way — 
thin,  regular  in  thickness,  not  torn,  not  compressed,  not 
rolled  or  curled,  and  not  folded  or  undulating  in  surface — 
without  careful  attention  to  a  host  of  minute  details.  These 
details  concern  (a)  the  position  of  the  knife,  and  (b)  the 
.consistency  (and  in  a  minor  degree  the  shape  and  position) 
of  the  mass  to  be.  cut. 

(a)  The  knife-position  may  be  considered  under  two  heads, 
viz.  its  slant  and  its  tilt. 

By  the  slant  of  the  knife  is  meant  the  angle  that  its  edge 
.makes  with  the  line  of  section  :  that  is,  with  the  line  along 
which  it  is  drawn  through  the  object  (or  along  which  the 
object  moves  across  it  in  the  case  of  microtomes  with  fixed 
knives).  The  position  is  transverse  when  the  edge  makes  an 
angle  of  90°  with  the  line  of  section,  or  the  knife  in  that 
case  is  said  to  beset  square.  It  is  oblique  or  slanting  when 
it  makes  a  smaller  angle  with  that  line. 

It  is  a  mistake  to  suppose  that  these  two  positions  differ  in  that  in  the 
transverse  position  the  knife  acts  as  a  wedge  or  ploughshare,  forcing  its 
way  straight  through  the  object,  whilst  in  the  oblique  position  it  acts  as  a 
saw,  its  edge  being  drawn  along  through  the  object,  as  in  free-hand  cut- 
ting. On  the  contrary,  in  both  cases  the  knife  acts  merely  as  a  wedge,  and 
no  microtome  in  general  use  at  the  present  time*  affords  a  drawing  move- 
ment such  as  can  be  given  by  the  hand.  In  either  position  of  the  knife 
no  point  of  the  object  is  ever  touched  by  more  than  one  point  of  the 
cutting-edge.  The  difference  between  the  effect  of  the  two  positions  is 

*  A  microtome  with  drawing  motion  to  the  knife  is  described  by  BECK 
in  Zeit.f.  wiss.  Mik.,  xiv,  3,  1897,  p.  324. 


IMBEDDING   METHODS    (PARAFFIN).  105 

merely  that  the  oblique  position  affords  a  more  acute-angled  wedge  than 
the  transverse  one. 

It  does  so  for  the  following  reason: — Neglecting  for  the  moment  the 
distinction  between  the  cutting-facets  and  the  surfaces  of  the  blade  (which 
are  distinct  usually  because  they  are  not  ground  to  the  same  angle),*  it  is 
clear  that  the  knife  itself  is  a  wedge,  the  angle  of  which  depends  on  the 
relation  between  the  height  of  its  base  and  the  distance  from  the  base  to  the 
edge.  With  the  same  base  the  angle  becomes  more  acute  the  greater  the  dis- 
tance from  edge  to  base.  Now  by  slanting  the  knife  we  can  effect  what  is 
equivalent  to  an  increase  in  the  distance  from  edge  to  base;  for  we  can  thus 
increase  the  distance  between  the  point  of  the  edge  which  first  touches  the 
object,  and  the  point  of  the  back  (strictly,  of  the  back  edge  of  the  under 
cutting-facet)  which  last  leaves  it.  When  the  knife  is  set  transversely,  the 
line  along  which  any  point  of  it  traverses  the  object  is  the  shortest  possible 
from  edge  to  base  of  the  wedge,  and  the  effective  angle  of  wedge  is  the  least 
acute  obtainable  with  that  knife.  But  if  it  is  set  as  obliquely  as  possible, 
the  line  along  which  any  point  of  it  traverses  the  object  traverses  the  knife 
from  heel  to  toe,  that  is,  along  the  greatest  possible  distance  from  edge  to 
base,  and  therefore  affords  practically  a  much  more  acute-angled  wedge 
than  in  the  first  case ;  and  so  on,  of  course,  for  intermediate  positions. 
(See  the  stereometrical  constructions  of  these  relations  by  SCHIEFFERDECKER, 
op.  cit.,  p.  115  ;  and  also,  with  more  instructive  figures,  APATHY,  "  Ueber 
die  Bedeutung  des  Messerhalters  in  der  Mikrotomie,"  in  Sitzberg.  med.- 
natunv.  Section  d.  Siebenbiirgischen  Museumvereins,  Bd.  xix,  Heft  7, 
p.  1  (Kolozsvar,  1897,  A.  K.  Ajtai). 

Very  large^  objects  are  best  cut  with  the  slanting  knife,  and  so  are  all 
objects  of  very  heterogeneous  consistency,  such  as  tissues  that  contain  much 
chitin  or  much  muscular  tissue.  In  general  all  very  difficult  objects  are 
better  cut  with  the  slanting  knife  than  the  transverse  one  (and  better  with 
a  slowly-working  sliding  microtome  than  with  a  quick-working  Rocker  or 
the  like).  Soft  masses,  such  as  gelatin  or  celloidin  cut  wet,  canjmly  be 
cut  with  the  slanting  knife.  The  slanting  position  causes  less  compression 
of  sections  than  the  transverse  one.  It  has  the  defect  of  producing  rolling 
in  paraffin  sections  more  easily  than  the  transverse  position.  The  letter  is 
the  proper  position  for  cutting  ribbons  of  sections  from  paraffin. 

By  the  tilt  of  the  knife  is  meant  the  angle  that  a  plane 
passing  through  its  back  and  edge  makes  with  the  plane  of 
section  :  or,  practically,  the  greater  or  less  degree  of  eleva- 
tion of  the  back  above  the  edge  (it  is  not  to  be  confounded 
with  the  inclination  of  the  long  axis  of  the  knife  to  the 

*  The  edge  of  a  microtome  knife  is  composed  of  two  plane  surfaces — the 
upper  and  lower  cutting-facets,  which  meet  one  another  at  an  acute  angle, 
the  cutting-edge,  and  posteriorly  join  on  to  the  upper  and  lower  surfaces  of 
the  blade  (see  some  good  figures  of  differently  shaped  knives  in  BEHBENS, 
KOSSEL  und  SCHIEFFERDECKER,  Das  Mikroskop.,  p.  115,  et  seq. ;  and  in 
APATHY'S  paper  quoted  below).  It  will  be  seen  that  the  two  facets 
together  form  a  wedge  welded  on  to  the  blade  by  the  base. 


106  CHAPTER    VIII. 

horizon;  any  accidental  inclination  that  this  may  have  is   a 
matter  of  no  moment). 

The  question  of  the  proper  tilt  to  be  given  to  the  knife 
under  different  circumstances  has  been  investigated  by 
APATHY,  luc.  cit.  supra.  He  concludes — (1)  The  knife  should 
always  be  tilted  somewhat  more  than  enough  to  bring 
the  back  of  the  under  cutting-facet  clear  of  the  object. 
(2)  It  should  in  general  be  less  tilted  for  hard  and  brittle 
objects  than  for  soft  ones ;  therefore,  caster  Is  paribus,  less  for 
paraffin  than  for  celloidin.  (3)  The  extent  of  useful  tilt 
varies  between  0°  and  16°  or  occasionally  20°.  (4)  Exces- 
sive tilt  causes  rifts  (longitudinal)  in  the  paraffin,  also 
furrows  that  in  bad  cases  split  up  the  section  into  narrow 
riBtons.  It  also  makes  sections  roll.  Also  it  may  cause 
the  knife  not  to  bite,  thus  causing  sections  to  be  missed.  Or 
it  may  give  an  undulatory  surface  to  the  sections,  owing  to 
vibrations  set  up  in  the  knife,  which  may  be  heard  as  a  deep 
humming  tone.  Further,  I  would  add,  excessive  tilt  may 
cause  the  knife  to  act  as  a  scraper,  carrying  «way  portions 
of  tissue  bodily  from  their  places.  Excessive  tilt  may  often 
be  recognised  by  the  knife  giving  out  a  short  metallic  sound 
just  as  it  leaves  the  object.  For  knives  with  plane  under 
surfaces  it  is  seldom  advisable  to  give  less  than  10  degrees 
tilt.  Knives  with  concave  under  surfaces,  on  the  contrary, 
may  require  to  be  placed  almost  horizontal.  Jung's  knife- 
holders  give  mostly  a  tilt  of  about  9°,  which  is  only  enough 
for  cutting  ribbons  with  hard  paraffin. 

A  knife  with  too  little  tilt  will  often  cut  a  second  section, 
or  fragments  of  one,  without  the  object  being  raised,  showing 
that  during  the  first  cut  the  object  was  pressed  down  by  the 
knife,  and  recovered  itself  afterwards.  This  fault  is  denoted 
by  the  ringing  tone  given  out  by  the  knife  on  passing  back 
over  the  object  before  the  latter  is  raised.  Such  a  knife 
gives  out  a  dull  rattling  sound  whilst  cutting.  Too  little 
tilt  causes  folding  or  puckering  of  sections,  and  does  not 
allow  of  the  cutting  of  the  thinnest  possible  sections,  as  the 
edge  does  not  bite  enough. 

Ribbon  section-cutting  requires  a  relatively  hard  paraffin 
and  less  tilt.  With  celloidin  it  is  very  important  to  avoid 
insufficient  tilt,  as  the  elastic  celloidin  yields  before  an  in- 
sufficiently tilted  knife  and  is  not  cut. 


IMBEDDING    METHODS    (PARAFFIN).  107 

The  tilt  of  the  knife  is  regulated  by  means  of  mechanical 
contrivances,  of  which  the  most  simple  are  the  horseshoe- 
shaped  wedges  of  NEUMAYER  (see  Jung's  price-list).  A  pair 
of  these,  each  ground  to  the  same  angle,  is  taken,  and  one 
of  them  placed  under,  and  the  other  over,  the  clamping-arm 
of  the  knife-holder.  Three  pairs,  having  different  degrees 
of  pitch,  are  supplied,  and  are  sufficient  for  most  work. 
Other  contrivances  to  the  same  end  consist  of  knife-holders 
that  permit  of  rotating  the  knife  on  its  long  axis,  and,  though 
more  costly,  will  be  found  a  great  convenience  where  much 
section-cutting  has  to  be  done.  For  these  see  Jung's  price- 
list,  and  various  recent  papers  in  the  Zeit.  f.  wis*.  Mik.,  also 
that  of  APATHY,  in  the  paper  quoted  above  (very  complicated), 
and  especially  the  description  of  the  two  latest  of  Jung,  viz. 
his  model  I  and  model  n,  by  MAYER  and  SCHOEBEL,  in  Zeit. 
f.  wiss.  Mik.,  xvi,  1899,  p.  29  (see  figure  of  model  I  in 
Journ.  Ifmj.  Mic.  Soc.,  132,  1899,  p.  o4b). 

(b)  As  to  the  consistency  and  shape  and  orientation  of 
the  mass  to  be  cut.  Solid  paraffin  varies  enormously  in  hard- 
ness according  to  the  temperature  of  its  surroundings.  It 
must  therefore  be  taken  of  a  melting-point  suitable  to  the 
temperature  of  the  laboratory,  harder  (i.  e.  of  higher  melting- 
point)  in  summer,  softer  in  winter.  On  this  point  see  below, 
§  141. 

As  to  the  shape  and  orientation  to  be  given  to  the  block 
to  be  cut,  these  differ  accordingly  as  the  cutting  is  done  with 
a  slanting  knife  or  a  square- set  knife.      In  the  first  case,  the 
block   is   best    trimmed  to   a  three-sided 
prism,  and  orientated  as  in  Fig.  4,  so  that 
the   knife  enters   it   at   the  angle  a   and 
leaves    it   at   the    angle    c.       When   the 
section  is  cut  it  will  adhere  to  the  knife 
only  by   the  angle   c,  and  can  thus  most 
readily  be  removed  by  means  of  a  brush 
or    needle.       The    object    itself    should 
come  to  lie  in  the  block  close  to  the  line 
b  c,    so  that  the  knife  at  first   cuts  only 
paraffin,  and  that  if  the  section  begins  to 
roll  it  may  be  caught  and  held   down  by 

a  brush  or  section-stretcher  before  the  object  itself  is  reached. 
For  the  square-set  knife  the  block  is  best  trimmed  to  a  four- 


108    .  CHAPTER  VIII. 

sided  prism,  and  orientated  as  in  the  first  case^  so  that  the 
knife  first  touches  one  ang'le,  if  the  sections  are  to  be  cut 
singly.  But  if  ribbons  are  to  be  cut,  the  block  must  be 
orientated  with  one  of  its  sides  parallel  to  the  knife-edge, 
and  the  opposite  side  must  be  strictly  parallel  to  this  one. 

For  NOACK'S  simple  apparatus  for  accurately  orientating 
small  blocks,  see  Zeit.  f.  wiss.  Alik.  xv,  1899,  p.  438,  or 
Journ.  Roy.  Mic.  Soc.,  132,  1899,  p.  550. 

For  ETERNOD'S  machine  for  trimming  blocks  to  true  cubes,  see  Zeit.f. 
wiss.  Mlik.,  xv,  4,  p.  421,  or  Journ.  Roy.  Mic.  Soc.,  1899,  p.  450. 

135.  Cutting  and  Section-stretching. — Paraffin  sections  are 
cut  dry — that  is,  with  a  knife  not  moistened  with  alcohol  or 
other  liquid.  By  this  means  better  sections  are  obtained,  but 
a  difficulty  generally  arises  owing  to  the  tendency  of  sections 
so  cut  to  curl  up  on  the  blade  of  the  knife.  It  is  sometimes 
difficult  by  any  means  to  unroll  a  thin  section  that  has  curled. 
To  prevent  sections  from  rolling,  the  following  points  should 
be  attended  to. 

First  and  foremost,  the  paraffin  must  not  be^top_Jiard,  but 
must  be  taken  of  a  melting-point  suitable  to  the  temperature 
of  the  laboratory,  see  §  111. 

The  exact  degree  of  hardness  necessary  must  be  deter- 
mined by  experiment.  If,  after  cutting  has  begun,  the 
paraffin  be  found  to  be  too  hard,  it  may  be  softened  by 
placing  a  lamp,  or  any  convenient  source  of  heat,  near  the 
imbedded  object.  I  find  that  a  mere  spirit  lamp  set  up 
near  the  object  will  sometimes  bring  the  paraffin  to  the 
right  consistency  in  a  few  minutes.  But  then,  the  paraffin 
being  warmed  most  on  the  side  nearest  the  lamp,  becomes 
softer  on  that  side,  and  the  sections  have  a  tendency  to 
become  compressed  and  puckered-in  on  that  side. 

If,  on  the  contrary,  the  paraffin  be  found  too  soft,  it  may 
be  hardened  by  exposing  it  to  the  cooling  influence  of  a  lump 
of  ice  placed  in  the  focus  of  a  parabolic  reflector. 

It  is  often  sufficient  to  moderate  the  temperature  of  the 
room  by  opening  or  closing  the  window,  stirring  the  fire, 
setting  up  a  screen,  or  the  like. 

Secondly,  the  knife  should  be  set  square,  for  the  oblique 
position  encourages  rolling,  and  the  more  the  knife  is  oblique 
the  more  do  the  sections  roll.  Not  that  a  square-set  knife 


IMBEDDING    ME1HUDS    (PAHAFFJN).  109 

will  always  cure  rolling  !  But  it  diminishes  the  tendency 
to  roll. 

Thirdly,  it  is  better  to  cut  ribbons  than  disconnected  sec- 
tions ;  ribbons  of  sections  will  often  cut  perfectly  flat,  even 
when  the  same  mass  will  only  give  rolled  sections  if  cut  dis- 
connectedly. For  if  a  section  has  only  a  slight  tendency  to 
curl,  it  will  be  held  down  flat  by  adhesion  to  the  one 
preceding  it. 

Special  masses  having  less  tendency  to  roll  than  pure 
paraffin  have  been  proposed.  Thus  a  mass  composed  of  four 
parts  of  hard  paraffin  and  one  of  vaselin  has  been  recom- 
mended. I  recommend,  however,  that  all  such  mixtures  be 
avoided. 

Mechanical  means  may  be  employed.  The  simplest  of 
these  is  as  follows  : 

During  the  cutting  the  edge  of  the  section  that  begins  to 
curl  is  caught  and  held  down  on  the  blade  of  the  knife  by 
means  of  a  small  camel-hair  brush  with  a  flat  point,  or  by  a 
small  spatula  made  by  running  a  piece  of  paper  on  to  the 
back  of  a  scalpel.  Or  the  section  is  held  down  by  means  of 
an  instrument  called  a  "  section-stretcher/'  This  consists 
essentially  of  a  little  metallic  roller  suspended  over  the  object 
to  be  cut  in  such  a  way  as  to  rest  on  its  free  surface  with  a 
pressure  that  can  be  delicately  regulated  so  as  to  be  sufficient 
to  keep  the  section  flat  without  in  any  way  hindering  the 
knife  from  gliding  beneath  it. 

See  the  descriptions  of  various  forms  of  section-stretchers,  Zool.  Anzeig., 
vol.  vi,  1883,  p.  100  (SCHTJLTZE)  ;  Mitth.  Zool.  Stat.  Neapel,  iv,  1883,  p.  429 
(MATEE,  ANDEES,  and  GIESBBECHT)  ;  Arch.f.  mik.  Anat.,  xxiii,  1884,  p.  537 
(DECKEB)  ;  Bull.  Soc.  Bdg.  Mic.,  x,  1883,  p.  55  (FBANCOTTE)  ;  The  Micro- 
scope, February,  1884  (GAGE  and  SMITH)  ;  WHITMAN'S  Meth.  in  Mic.  Anat.* 
1885,  p.  91 ;  Zeit.f.  wiss.  Mik.,  iv,  2, 1887,  p.  218  (STEASSEE)  ;  Zeit.f.  wiss. 
Mik.,  x,  2,  1893,  p.  157,  or  Journ.  Boy.  Mic.  Soc.,  1894,  p.  132  (Bosx) ;  as 
well  as  Journ.  Roy.  Mic.  Soc.,  iii,  pp.  450,  916,  and  other  places.  It  must 
be  allowed  that  all  these  instruments  are  difficult  to  use,  and  that  if  they 
are  not  perfectly  adjusted  they  may  easily  injure  the  sections.  And  they 
are  less  necessary  than  formerly,  now  that  good  processes  for  flattening  out 
sections  have  been  worked  out  (see  below,  "  Section-flattening,"  §  138). 

Another  plan  is  to  allow  the  sections  to  roll,  but  to  control 
the  rolling.  To  this  end,  the  block  of  paraffin  is  pared  to  the 
shape  of  a  wedge  five  or  six  times  as  long  as  broad,  the 
object  being  contained  in  the  broad  part,  and  the  edge 


110  CHAPTER    VIII. 

turned  towards  the  knife  (see  Fig.  4).  The  sections  are 
allowed  to  roll  and  come  off  as  coils,  the  section  of  the  object 
lying  in  the  outermost  coil,  which  will  be  found  to  be  a  very 
open  one — indeed,  very  nearly  flat.  Lay  the  coil  on  a  slide 
with  this  end  downwards,  warm  gently,  and  the  part 
containing  the  object  will  unroll  completely  and  lie  quite 
flat. 

A  defect  opposite  to  that  of  the  rolling  of  sections  is  the 
compression  and  the  crumpling  or  puckering  of  sections, 
indicating  that  the  paraffin  Eas  been  compressed  by  the  knife 
instead  of  being  merely  cut  true  by  it.  Such  sections, 
besides  showing  creases  or  folds,  have  a  smaller  area  than 
that  of  the  block  from  which  they  are  cut.  This  is  a  bad 
fault,  for  the  compression  may  obliterate  important  cavities 
or  efface  important  limits  between  cell-layers^  etc.  It  may 
be  caused  by  a  badly-cutting  knife,  and  is  very  easily  caused 
l>y  the  paraffin  being  too  soft.  To  prevent  it,  correct  the 
knife  or  cool  the  paraffin,  or  re-imbed  in  harder  paraffin. 
If  the  crumpling  has  not  gone  so  far  as  to  cause  the  folds  of 
the  sections  to  adhere  to  one  another,  the  sections  may  be 
perfectly  cured  by  flattening  on  water ;  see  below,  ' '  Section- 
flattening/'  §  138. 

Devices  for  heating  or  for  cooling  the  knife,  with  a  view  to  the  improve- 
ment of  cutting,  have  been  described  ;  see  VAN  WALSEM  in  Zeit.f.  wiss.  Milt., 
xi,  2,  1894,  p.  218  ;  also  Jung's  price-list.  I  have  myself  sometimes  found 
it  advantageous  to  warm  the  knife. 

136.  Collodionisation  of  Sections. — Some  objects  are  by 
nature  so  brittle  that,  notwithstanding  all  precautions  taken 
in  imbedding  and  previous  preparation,  they  break  or  crumble 
before  the  knife,  or  furnish  sections  so  friable  that  it  is 
impossible  to  mount  them  in  the  ordinary  way  without  some 
impairment  of  their  integrity.  Ova  are  frequently  in  this 
case.  The  remedy  for  this  state  of  things  consists  in  cover- 
ing the  exposed  surface  of  the  object  just  before  cutting  each 
section  with  a  thin  layer  of  collodion,  which  serves  to  hold 
together  the  loose  parts  of  even  the  most  fragile  sections  in 
a  wonderfully  efficacious  way ;  and  the  same  treatment  ap- 
plied to  tissues  which  are  not  specially  fragile  will  enable  the 
operator  to  cut  sections  considerably  thinner  than  can  be 
obtained  in  the  usual  way.  BUTSCHLI  has  obtained  in  this 
manner  sections  of  less  than  1  /m  in  thickness. 


IMBKDD1NG    MKTHODS    (PARAFFJN).  Ill 

The  primitive  form  of  the  process  was  to  place  a  drop  of 
collodion  on  the  free  surface  of  each  section  just  before 
rutting  it.  But  this  practice  has  two  defects  ;  the  quantity 
of  collodion  employed  sensibly  softens  the  paraffin,  and  the 
thick  layer  of  collodion  when  dry  causes  the  sections  to  roll. 

MAKK  (Amer  Natural.,  1885,  p.  628;  cf.  Joum.  R(,y.  Mic. 
Soc.,  1885,  p.  738)  gives  the  following  directions  : 

"  Have  ready  a  little  very  fluid  collodion  in  a  small  bottle, 
through  the  cork  of  which  passes  a  small  camel- hair  brush, 
which  just  dips  into  the  collodion  with  its  tip.  The  collodion 
should  be  of  such  a  consistency  that  when  applied  in  a  thin 
layer  to  a  surface  of  paraffin  it  dries  in  two  or  three  seconds 
"without  leaving  a  shiny  surface.  Collodion  of  this  consistency 
does  not  produce  a  membrane  on  the  paraffin  in  drying,  and 
therefore  has  no  tendency  to  cause  sections  to  roll.  It  has 
further  the  advantage  that  it  penetrates  to  a  certain  depth 
below  the  surface  of  the  preparation,  and  fixes  the  deeper 
layers  of  it  in  their  places.  The  collodion  must  be  diluted 
with  ether  as  soon  as  it  begins  to  show  signs  of  leaving  a 
shiny  surface  on  the  paraffin. 

"  Take  the  brush  out  of  the  collodion,  wipe  it  against  the 
neck  of  the  bottle,  so  as  to  have  it  merely  moist  with  collodion, 
and  quickly  pass  it  over  the  free  surface  of  the  preparation. 
Care  must  be  taken  not  to  let  the  collodion  touch  the  vertical 
surfaces  of  the  paraffin,  especially  not  the  one  which  is  turned 
towards  the  operator,  as  that  will  probably  cause  the  section 
to  become  stuck  to  the  edge  or  under  surface  of  the  knife. 
As  soon  as  the  collodion  is  dry,  which  ought  to  be  in  two  or 
three  seconds,  cut  the  section,  withdraw  the  knife,  and  pass 
the  collodion  brush  over  the  newly-exposed  surface  of  the 
paraffin.  Whilst  this  last  layer  of  collodion  is  drying,  take 
up  the  section  from  the  knife  and  place  it  with  the  collodion- 
ised  surface  downwards  on  a  slide  prepared  with  fixative  of 
Schaellibaum.  Then  cut  the  second  section,  and  repeat  the 
manipulations  just  described  in  the  same  order.  A  skilful 
operator  can  cut  ribbons  of  sections,  collodionising  each 
section/' 

HENKING  (Zeit.  f.  wiss.  Mi~k.,  iii,  4,  1886,  p.  478)  objects  to 
the  above  process  that  the  ether  of  the  collodion  softens  the 
paraffin,  and  proposes  a  solution  of  paraffin  in  absolute  alcohol. 
The  solution  is  made  by  scraping  paraffin  into  absolute  alcohol. 


112  CHAPTER    VIII. 

For  extremely  brittle  objects,  such  as  ova  of  Phalangida, 
the  same  author  recommends  a  thin  (light  yellow)  solution  of 
shellac  in  absolute  alcohol. 

HEIDER  (Embryonalentw.  v.  Hydrophilus,  1889,  p.  12;  cf. 
Zeit.  f.  iviss.  Mik  ,  viii,  4,  1892,  p.  509)  employs  a  solution 
made  by  mixing  a  solution  of  gum  mastic  in  ether,  of  a 
syrupy  consistency,  with  an  equal  volume  of  collodion,  and 
diluting  the  mixture  with  ether  until  quite  thin  and  liquid. 

RABL  (ibid.,  xi,  2,  1894,  p.  170)  employs  superheated 
paraffin,  kept  at  a  temperature  of  about  100°  C.  on  a  water- 
bath.  This  plan  has  the  advantage  of  efficiently  filling  up 
any  cavities  there  may  be  in  the  objects,  and  also  of  prevent- 
ing the  sections  from  rolling. 

APATHY  (Mikrotechnik,  p.  183)  employs  a  1  per  cent, 
solution  of  celloidin,  allows  the  sections  to  roll,  and  unrolls 
them  by  the  water-process. 

137.  Ribbon  Section-cutting. — If  a  series  of  paraffin  sections 
be  cut  in  succession  and  not  removed  from  the  knife  one  by 
one  as  cut,  but  allowed  to  lie  undisturbed  on  the  blade,  it 
not  unfrequently  happens  that  they  adhere  to  one  another 
by  the  edges  so  as  to  form  a  chain  or  ribbon  which  may  be 
•  taken  up  and  transferred  to  a  slide  without  breaking  up, 
thus  greatly  lightening  the  labour  of  mounting  a  series.  The 
following  appear  to  me  to  be  the  factors  necessary  for  the 
production  of  a  ribbon. 

Firstly,  the  paraffin  must  be  of  a  melting-point  having  a 
certain  relation  to  the  temperature  of  the  laboratory.  I  find 
that  small  sections  can  always  be  made  to  chain  when  cut 
from  a  good  paraffin  of  45°  C.  melting-point  in  a  room  in 
which  the  thermometer  stands  at  16°  to  17°  C.,  and  that,  for 
the  Thoma  microtome,  at  15°  C.  the  paraffin  is  a  trifle  hard. 
But  see  on  this  point  §  141.  Secondly,  the  knife  should  be 
set  square.  Thirdly,  the  block  of  paraffin  should  be  pared 
down  very  close  to  the  object  and  should  be  cut  so  as  to 
present  a  straight  edge  parallel  to  the  knife  edge ;  and  the 
opposite  edge  should  also  be  parallel  to  this.  Fourthly,  the 
sections  ought  to  be  jyut  rapidly,  with  the  swiftest  strokes  ^ 
that  can  be  produced.  For  it  is  the  sharp  impact1  of  the 
knife  that  slightly  heats,  and  therefore  slightly  softens  the 
near  edge  of  the  paraffin,  and  thus  causes  the  sections  to 


IMBEDDING   METHODS   (  PARAFFIN).  113 

• 

cohere.  It  is  by  no  means  necessary  for  this  purpose  to  have 
recourse  to  special  mechanical  contrivances,  as  in  the  so-called 
ribbon  microtomes.  The  Thoma  microtome  well  flooded  with 
oil  is  sufficient.  But  the  automatic  microtomes,  and  amongst 
them  the  Cambridge  Rocking  Microtome,  the  Reinhold-Giltay, 
and  the  Minot,  are  certainly  most  advantageous  for  this  pur- 
pose. 

Various  plans,  such  as  coating  the  edges  of  the  paraffin  with  softer 
paraffin,  or  with  Canada  balsam,  or  the  employment  of  specially  prepared 
paraffin,  have  been  recommended,  with  the  idea  that  they  help  the  sections 
to  stick.  I  find  that  none  of  these  devices  are  Accessary.  For  the  prepared 
paraffin  of  Spee,  see  below,  §  142. 

MAYER,  however  (Grundzilge,  p.  86),  remarks  hereon,  that  though 
coating  with  a  softer  paraffin  is  not  necessary  when  soft  paraffin  is  taken i 
for  imbedding,  yet  if  a  paraffin  of  J>5  to  60  melting-point  is  used  for 
imbedding,  it  is  absolutely  necessary  to  coat  it  with  softer,  for  sections  of 
1 )  p.  thickness,  and  at  least  advisable  for  thinner  ones.  To  coat  the  block, 
take  paraffin  of  about  40°  C.  melting-point,  melt  it,  heat  it  to  about  80°  on 
the  water-bath,  dip  the  block  into  it  for  an  instant,  and  rapidly  turn  it  over 
so  that  the  fluid  paraffin  may  run  down  away  from  the  top  part  as  much  as 
possible.  Allow  it  to  cool,  and  pare  away  again  the  soft  paraffin  from  the 
two  sides  that  are  not  to  be  arranged  parallel  to  the  knife.  Large  blocks 
may  have  two  coatings  given  them. 

It  sometimes,  though  rarely,  happens  that  the  ribbon  becomes  electrified 
during  the  cutting,  and  twists  and  curls  about  in  the  air  in  a  most  fantastic 
and  undesirable  manner.  It  may  be  got  flat  by  warming  slightly  ;  but 
there  is  no  known  means  of  preventing  the  electrification. 

138.  Section-flattening  (very  important). — The  sections 
having  been  obtained  may  be  cleared  and  mounted  at  once 
if  they  are  quite  perfect,  that  is,  neither  rolled  nor  creased 
nor  compressed.  But  should  they  in  the  least  degree  show 
any  of  these  defects,  they  must  first  be  unrolled  or  smoothed, 
or  expanded  to  their  proper  dimensions.  It  is  most  impor- 
tant not  to  neglect  this  point,  as  is  often  done  in  the  case 
of  sections  that  are  neither  rolled  nor  crumpled,  but  are 
rrntijiressed,  as  shown  by  their  being  of  smaller  area  than 
the  block  from  which  they  have  been  cut. 

The  most  efficacious  plan  for  flattening  and  expanding 
sections  is  the  combined  treatment  with  fluid  and  heat 
(GASKELL,  Quart.  Journ.  Mic.  Sci.,  xxxi,  1890,  p.  382  ;  M. 
DUVAL,  Journ.  de  I'Anat.  et  de  la  PhysioL,  1891,  p.  26  ; 
HKXXEGUY,  ibid.,  1891,  p.  398;  GULLAND,  Journ.  of  Anat. 
<nul  PhysioL,  1891,  p.  56;  and  others).  The  sections  are 

8 


114  CHAPTER    VIII. 

• 

either  floated  on  to  the  surface  of  warm  water  or  warm 
alcohol  contained  in  a  watch  glass  or  suitable  dish,,  which 
causes  them  to  flatten  out  perfectly,  and  are  then  transferred 
to  a  slide,  by  floating  them  into  position,  or  otherwise.  Or 
the  slide  has  a  layer  of  water  spread  over  it,  the  sections 
are  laid  on  the  water,  and  the  slide  is  heated  (to  somewhat 
below  the  melting-point  of  the  paraffin)  until  the  sections 
flatten  out,  which  happens  in  a  few  seconds.  The  method 
can  be  made  available  for  fixing  series  of  sections  to  the 
slide  ;  the  further  details  necessary  for  the  successful  ac- 
complishment of  this  are  given  in  the  chapter  on  "  Serial 
Section  Mounting  "  (the  Water  Method). 

A  special  water-bath  for  flattening  sections  is  described  by  NOWAK  in 
.Zeit.f.  wiss.  Mik.,  xii,  4,  1896,  p.  447). 

VAN  WALSEM  (Zeit.f.  wiss.  Mik.t  xi,  2,  1894,  p.  228)  describes  a  plan 
according  to  which  the  sections  are  arranged  on  a  strip  of  parchment-paper 
which  is  moistened  and  passed  over  a  warmed  cylinder  revolving  in  water  on 
the  principle  of  a  postage-stamp  dampener  (see  abstract  with  illustration  in 
Journ.  Roy.  Mic.  8oc.,  1895,  p.  121). 

139.  Clearing  and  Mounting, — The  sections  having  been 
duly  smoothed  by  one  of  these  processes,  and  duly  fixed  to 
the  slide  (see  "  Serial  Section  Mounting ")  (unless  it  is 
desired  to  keep  them  loose)  all  that  now  remains  is  to  get 
rid  of  the  paraffin  and  mount  or  stain  as  the  case  may  be. 
Many  solvents  of  paraffin  have  been  recommended  for  free- 
ing sections  from  the  paraffin  with  which  they  are  infil- 
trated : — Turpentine,  warm  turpentine,  a  mixture  of  4  parts 
of  essence  of  turpentine  with  1  of  creasote,  creasote,  a 
mixture  of  turpentine  and  oil  of  cloves,  benzin,  toluol,  xylol, 
thin  solution  of  Canada  balsam  in  xylol  (only  applicable  to 
very  thin  sections),  hot  absolute  alcohol,  naphtha,  or  any 
other  paraffin  oil  of  low  boiling-point.  Of  these  xylol,  toluol, 
benzol,  and  chloroform  are  generally  in  most  respects  the 
best. 

If  the  slide  be  warmed  to  the  melting-point  of  the  paraffin, 
a  few  seconds  will  suffice  (with  thin  sections)  to  remove  the 
paraffin  if  the  slide  be  plunged  into  a  tube  of  xylol  or  toluol. 
The  sections  may  be  mounted  direct  from  the  xylol  or  the 
slide  may  be  brought  into  a  tube  of  alcohol  to  remove  the 
solvent  for  staining. 


1MBKDDING    MKTHODS    (PARAFFIN).  115 

140.  Recapitulation  of  the  Paraffin  Method,  as  recommended 
to  be  practised  for  small  objects. — Put  into  a  small  test-tube 
enough  oil  of  cedar  to  cover  your  object.      On  to   the  oil 
pour  carefully  the  same  quantity  of  absolute  alcohol.      Take 
your   (already  dehydrated)   object  and  put  it  carefully  into 
the  alcohol.      Leave  it  until  it  has  sunk  to  the  bottom  of  the 
cedar  oil.      Wait  till  the  refraction  lines,  §  106,  have  vanished. 
Then  put  it  into  paraffin  kept  at  melting-point  in  a  watch 
glass.      Let  the  paraffin  be  of  the  very  lowest  melting-point 
that  will  give  sufficiently  thin  sections,  and  to  this  end  work 
in  a  cool  place.      After  a  time  change  the  paraffin  (i.  e.  put 
the  object  into  a  fresh  watch  glass  with  clean  paraffin)  once,, 
or  twice  if  the  object  be  at  all  large.      As  soon  as  the  object, 
is  thoroughly  soaked  with  paraffin  float  the  watch  glass  on 
cold  water.      When  cool,  cut  out  a  block  of  paraffin  contain- 
ing the  object,  and  fix  it  with  a  heated  needle  on  a  cone  of 
paraffin  already  mounted  on  the  object- carrier  of  the  micro- 
tome. 

Trim  and  orient  the  block  and  knife  according  to  circum- 
stances, as  directed  under  6,  §  134.  Cut  the  sections,  singly 
if  desired,  or  for  convenience  in  ribbons.  Collodionise  if 
necessary.  When  cut  always  flatten  and  expand  on  water, 
§  138.  Fix  them  in  serial  order  on  a  slide  by  one  of  the 
methods  given  in*the  chapter  on  "  Serial  Section  Mounting/' 
the  water  method  by  preference  if  they  have  to  be  stained. 
Warm  and  remove  the  paraffin  with  xylol.  Stain,  or  mount 
directly. 

Paraffin  Masses. 

141.  Pure  Paraffin. — It  is  now  almost  universally  admitted 
that  pure  paraffin  forms  an  imbedding  mass  greatly  superior 
for  ordinary  work  to  any  of  the  many  mixtures  with  wax  and 
the  like  that  used  to  be  recommended.      A  paraffin  melting 
at  45°  C.  is   that  which   in    my  experience    gives   the   best 
results  so  long  as  the  temperature  of  the  laboratory  is  between 
15°  and  17°  C.  ;   whilst  for  a  temperature  of  22°  C.  a  paraffin 
melting  at  48°  is  required.      And  for  higher  temperatures  a 
still  harder  paraffin,  of  over  50°  melting-point,  is  required. 

Paraffin  of  various  melting-points  is  easily  found  in  com- 
merce. Intermediate  sorts  may  be  made  by  mixing  hard 


U6  CHAPTER   VIII. 

and  soft  paraffin.  Two  parts  of  paraffin  melting  at  50° 
with  one  of  paraffin  melting  at  36°  C.  give  a  mass  melting 
at  48°  C. 

Many  workers  of  undoubted  competence  prefer  masses 
somewhat  harder  than  those  recommended,  viz.  of  melting- 
points  varying  between  50°  and  55°  C.  for  the  normal  tem- 
perature of  the  laboratory;  and  others  recommend  masses 
melting  at  60°  C.  or  higher. 

So,  for  instance,  Heidenhain  (58°),  Apathy  (55°),  Eabl 
(56°),  Mayer  (58°  to  60°  in  summer;  in  winter  about  56°, 
but  never  less  than  50°).  Mayer  points  out  (Gruiidziige, 
p.  90)  that  at  Naples  the  temperature  during  five  months  of 
the  summer  and  autumn  is  over  22°  C.  in  the  laboratory, 
sometimes  over  30°.  Temperatures  such  as  these  are  seldom 
realised  in  the  British  Isles,  and  whilst  I  quite  admit  that 
the  hard  paraffin  employed  by  Mayer  may  have  its  raison 
d'etre  for  Naples,  I  hold  that  for  that  very  reason  it  is  in 
general  unnecessarily  hard  for  cooler  climates. 

For  thin  sections  a  harder  paraffin  is  required  than  for 
thick  ones ;  and  the  thinner  the  sections,  the  harder  should 
the  paraffin  be. 

The  figures  above  given  have  been  repeatedly  verified  and 
are  undoubtedly  correct.  But  an  important  explanation 
remains  to  be  made.  The  statements  refei*  to  work  with  the 
Thoma  sliding  microtome.  I  have  since  ascertained  that 
microtomes  with  fixed  knives,  such  as  the  Cambridge,  the 
Minot,  or  the  Reinhold-Giltay,  will  give  good  results,  so  far 
as  cutting  is  concerned,  with  much  harder  paraffin,  and,  in 
fact,  require  such.  This  is  an  advantage,  so  far  as  the 
obtaining  of  very  thin  sections  is  concerned  ;  but  it  seems 
to  me  to  remain  true  that  for  delicate  work  it  is  well  in  the 
interest  of  the  preservation  of  the  tissues  to  use  a  paraffin  of 
as  low  a  melting-point  as  possible. 

Paraffin  had  better  be  obtained  from  Griibler,  or  one  of 
the  known  dealers  in  microscopic  reagents.  BRASS  (Zeit.  f. 
wiss.  Mik.,  ii,  1885,  p.  300)  recommends  such  as  has  been 
kept  for  some  years,  as  it  has  less  tendency  to  crystallise 
than  new  paraffin. 

124.  Prepared  Paraffin  (Pure). — G-RAF  SPEE  (Zeit.  f.  wiss. 
Milf.,  ii,  1885,  p.  8)  recommends  the  following  preparation 


IMBEDDING    METHODS    (PARAFFIN).  117 

of  commercial  paraffin  as  giving  a  mass  particularly  favour- 
able for  ribbon- section  cutting.  Paraffin  of  about  50°  C. 
melting-point  is  taken  and  heated  in  a  porcelain  capsule  by 
means  of  a  spirit  lamp.  After  a  time  disagreeable  white 
vapours  are  given  off,  and  the  mass  shrinks  a  little.  This 
result  is  arrived  at  in  from  one  to  six  hours,  according  to 
the  quality  of  the  paraffin.  The  mass  then  becomes  brownish- 
yellow,  and  after  cooling  shows  an  unctuous  or  soapy  surface 
on  being  cut.  The  melting-point  will  be  found  to  have  risen 
several  degrees.  This  mass  may  be  obtained  ready  prepared 
from  Griibler.  The  object  of  this  preparation  is  to  make  the 
mass  stickier,  in  view  of  cutting  ribbons. 

For  mixtures  of  paraffin  with  other  substances,  none  of  which  I  consider 
to  offer  any  advantage,  see  previous  editions. 

143.  Soap  Masses. — These  have  never  been  much  used,  and  are  now  en- 
tirely discarded  in  favour  of  paraffin.  But  see  previous  editions,  or  the  papers 
of  POLZAM  (MorpTi.  Jahrb.,  iii,  1877,  3tes  Heft,  p.  558,  from  Salensky's 
paper  on  the  gemmation  of  Salpa,  loc.  cit.) ;  KADYI  (Zool.  Anz.,  37,  1879, 
vol.  ii,  p.  477) ;  DOLLKEN  (Zeit.f.  wiss.  Mik.,  xiv,  1, 1897,  p.  32). 


Gelatin  Masses. 

144.  Gelatin  Imbedding  is  a  method  that  has  the  advantage 
of  being  applicable  to  tissues  that  have  not  been  in  the  least 
degree  dehydrated,  and  may  render  great  service  in  the 
study  of  very  watery  objects. 

The  modus  operandi  is,  on  the  whole,  the  same  as  for  other 
fusion  masses,  with  the  difference  that  the  objects  are  pre- 
pared by  saturation  with  water  instead  of  alcohol  or  a  clearing 
agent.  After  the  cooling  of  the  mass  it  may  sometimes  be 
cut  at  once,  but  it  is  generally  necessary  to  harden  it.  This 
may  be  done  by  treatment  for  a  few  minutes  with  absolute 
alcohol  (KAISER),  or  for  a  few  days  with  90  per  cent,  alcohol 
(KLEBS)  or  chromic  acid  (KLEES)  or  formaldehyde  (Ni COLAS), 
or  it  may  be  frozen  (SoiLAs). 

The  mass  is  removed  from  the  sections  by  means  of  warm 
water. 

MAYEB  (Mitth.  Zool  StaL  Neapel,  ii,  1880,  p.  27)  for  coarse  researches 
leaves  the  mass  in  the  sections  (if  the  tissues  have  been  stained  previously) 
as  it  becomes  sufficiently  transparent  in  balsam.  He  recommends  as  a 


118  CHAPTKB    VIII. 

convenient  support  on  which  to  gum  the  imbedded  objects  with  warm 
gelatin  his  artificial  pith,  thus  made  :  gelatin  swollen  in  water  is  thoroughly 
shaken  up  with  5  to  -|  its  volume  of  castor  oil,  and  the  mixture  poured  into 
a  capsule  when  just  on  the  point  of  cooling;  the  castor  oil  being  then 
extracted  bj  means  of  90  per  cent,  alcohol,  the  gelatin  remains  behind  as  a 
finely-porous  inelastic  mass. 

APATHY  (ibid.,  xii,  1897,  p.  718)  saturates  objects  with  thin  glycerin- 
gelatin,  allows  the  water  to  evaporate  from  it  in  a  desiccator  kept  just  at 
the  melting  temperature  of  the  mass,  imbeds  in  metal  squares,  §  126, 
hardens  in  absolute  alcohol,  and  cuts  under  the  same. 

145.  KLEBS'  Gelatin  (Glycerin  Jelly)  (Arch.  f.  mik.  Anat., 
v,  1869,  p.  165). — A  concentrated  solution  of  isinglass  is 
mixed  with  half  its  volume  of  glycerin. 


146.  KAISER'S  Gelatin  (Bot.  Centralb.,  i,  1880,  p.  25  ;  Journ. 
Roy.  Mic.  Soc.,  iii,  1880,,  p.  504). — One  part  by  weight  of  the 
finest  French  gelatin  is  left  for  about  two  hours  in  6  parts  by 
weight  of    water;    7  parts  of  glycerin  are  added,   and   for 
every  100  grins,  of  the  mixture  1  grm.  of  concentrated  car- 
bolic acid.      The  whole  is  warmed  for  ten  to  fifteen  minutes, 
stirring  all  the  while,  until  the  whole  of  the  flakes  produced 
by  the  carbolic  acid  have  disappeared.      Filter  whilst  warm 
through  the  finest  spun    glass,  which   has   been   previously 
washed  in  water  and  laid  whilst  wet  in  the  funnel. 

147.  GERLACH'S  Gelatin  (GKRLACH,  Unters.  a.  d.  Anat.  Innsl. 
Erlangen,  1884;   Journ.  Hoy.  Mic.  Soc.,  1885,  p.  541). — Take 
gelatin,  40  grms. ;  saturated  solution  of.  arsenious  acid,  200 
c.c.  ;    glycerin,    J20  c.c.      Clarify   with  white  of  egg.      The 
mass  may  be  kept  for  years  in  a  well-stoppered  bottle.      The 
objects  to  be  prepared  for  imbedding  by  a  bath  of  one  third 
glycerin. 

148.  BKONOTTI'S  Gold  Gelatin  Mass   (Journ.  de  Botan.,  vi, 
1892,  p/194;  Journ.  fioy.  Mic.  Soc.,  1892,  p.  706).— Twenty 
grms.  gelatin  dissolved  with  heat  in  200  c.c.  distilled  water, 
and  30  to  40  c.c.  of  glacial  acetic  acid  with  1  grm.  corrosive 
sublimate  added  after  filtering.     At  the  temperature  of  15°  C. 
the  mass  has  the  consistence  of  a  thick  syrup.      Objects  are 
prepared  by  soaking  in  some  of  the  mass   diluted  with  two 
to  three  vols.  of  water,  then  imbedded  in  the  undiluted  mass. 


IMBEDDING    METHODS    (GELATIN). 

The  mass  is  then  hardened  in  spirit  or  bichromate  of  potash, 
picric  acid,  or  the  like.  No  heat  at  all  is  required  in  this 
process. 

149.  NICOLAS'S  Method  (Bibliogr.  Anat.,  Paris,  3  annee, 
I  1896,  p.  274;  Zeit.f.  wiss.  Mik.,  xiii,  1896,  p.  218).— Pre- 
parations are  first  soaked  for  one  or  two  days  in  a  3  per  cent, 
to  4  per  cent,  aqueous  solution  of  gelatin  kept  at  25°  C., 
then  for  the  same  time  in  a  10  per  cent,  solution,  and  then 
[  for  two  or  three  days  more  in  a  20  per  cent,  to  25  per  cent, 
solution  containing  8  per  cent,  to  10  per  cent,  of  glycerin 
and  kept  at  35°  C.  They  are  then  imbedded  in  some  of  the 
same  mass  in  paper  trays,  and  as  soon  as  the  gelatin  has  set 
are  thrown  into  a  5  per  cent,  solution  of  formaldehyde 
(formol  1  part,  water  7).  After  a  few  days  therein  the 
gelatin  has  become  hard  and  insoluble,  and  may  be  cut  or 
preserved  for  months  in  weak  formol  solution,  or  dilute 
alcohol  or  glycerin,  or  even  in  pure  water.  The  mass  cuts 
like  celloidin,  but  unfortunately  takes  stains  strongly.  The 
sections  must  be  very  carefully  and  gradually  passed  through 
the  successive  alcohols  for  dehydration,  as  they  curl  up  very 
easily.  They,  however,  flatten  out  at  once  on  being  brought 
from  absolute  alcohol  into  cresylol,  and  may  thence  be 
mounted  in  balsam.  To  mount  in  glycerin  is  of  course  easy. 


CHAPTER    IX. 
COLLODION  (CELLOIDIN)  AND  OTHER  IMBEDDING  METHODS. 

150.  Advantages  of  the  Collodion  or  Celloidin  Method. — Collo- 
dion (or  celloidin)  masses  do  not  require  the  employment  of 
heat.  They  do  not  require  that  the  objects  should  be  cleared 
before  imbedding,  and  that  is  an  advantage  in  the  case  of 
very  large  objects.  They  are  more  or  less  transparent, 
which  facilitates  the  orientation  of  the  object.  And  they 
are  specially  indicated  for  very  large  objects,  for  the  soaking 
in  collodion  being  quite  inoffensive  to  the  most  delicate  ele- 
ments may  be  prolonged  if  necessary  for  weeks,  thus  ensuring 
the  harmless  penetration  of  objects  that  would  be  literally 
cooked  if  they  were  submitted  to  a  paraffin  bath  of  like 
duration.  Lastly,  the  mass  being  quite  transparent  after 
mounting,  it  is  not  necessary  to  remove  it  from  the  sections 
before  staining  and  mounting  them ;  it  may  remain,  and 
fulfil  the  function  of  an  admirable  support  to  the  tissues, 
holding  in  their  places  brittle  or  detached  elements  that 
without  that  help  would  fall  to  pieces  and  be  lost. 

There  are  two  disadvantages.  One  is  that  the  process  is 
a  very  long  one ;  as  usually  practised,  the  collodion  process 
requires  some  three  days  for  the  imbedding  of  an  object  that 
can  be  imbedded  in  paraffin  in  an  hour  (though  the  time 
may  be  greatly  abridged  by  GILSON'S  rapid  process  given 
below).  Another  is  that  it  is  impossible  to  obtain  with 
celloidin  sections  as  thin  as  those  furnished  by  paraffin  ;  the 
lowest  limit  I  have  been  able  to  attain  to  is  7  ju,  which  for 
some  work  is  not  sufficient.  Other  workers  seem  to  have 
obtained  thinner  ones ;  but  at  any  rate  this  cannot  be  done 
without  difficulty. 

As  to  the  choice  of  a  process,  I  urgently  recommend   the 


COLLODION    AND    OTHER    IMBEDDING   METHODS.         121 

recently   introduced  practice   of  clearing  before  cutting,  and 
cutting  dry  as  described  in  §  165. 

151.  Collodion,    Celloidin,    and    Photoxylin. — The    collodion 
method  is  due  to  DDVAL  (Journ.  de  VAnat.,  1879,  p.  185). 

Celloidin,  recommended  later  on  by  MERKEL  and  SCHIEFFER- 
DECKER  (Arch.  f.  Anat.  u.  Phys.,  1882,  p.  200),  is  merely  a 
patent  collodion.  It  may  be  obtained  from  GRUBLER,  or  the 
other  dealers  in  histological  reagents.  It  is  sent  out  in  the 
form  of  tablets  of  a  tough  gelatinous  consistency  and  slightly 
milky- white  transparency.  These  tablets  may,  if  desired,  be 
dissolved  at  once  in  ether,  or  a  mixture  of  ether  and  alcohol, 
to  make  a  collodion  of  any  desired  strength.  But  it  is 
better,  as  recommended  by  APATHY,  to  cut  them  up  into  thin 
shavings,  which  should  be  allowed  to  dry  in  the  air  until  they 
become  yellow,  transparent,  and  of  a  horny  consistency,  and 
that  these  be  then  dissolved  in  alcohol  and  ether  (sulphuric, 
free  from  acid) .  The  solutions  thus  prepared  are  free  from 
the  excess  of  water  that  is  present  in  the  undried  celloidin, 
and  give  after  hardening  a  mass  that  is  more  transparent  and 
of  a  better  consistency  for  cutting  (Zeit.  f.  iviss.  Mik.,  vi,  2, 
1889,  p.  1(34). 

Imbedding  masses  of  excellent  quality  can  be  prepared 
with  ordinary  collodion,  but  celloidin  furnishes  more  readily 
solutions  of  known  concentration.  Otherwise  there  is  but 
little  to  choose  between  the  two,  and  therefore  in  this  work 
the  terms  collodion  and  celloidin  are  used  indifferently. 

Photoxylin  (KBYSINSKY,  VIBCHOW'S  Archiv,  cviii,  1887,  p.  217  ;  BUSSE, 
Zeit.  f.  wiss.  Mile.,  ix,  1,  1892,  p.  47)  is  a  dry  substance,  of  the  aspect  of 
cotton  wool,  and  chemically  nearly  related  to  celloidin.  It  can  be  obtained 
from  GRUBLER.  It  gives  a  clear  solution  in  a  mixture  of  equal  parts  of 
ether  and  absolute  alcohol,  and  should  be  used  in  exactly  the  same  way  as 
celloidin.  It  has  the  advantage  of  affording  a  mass  which  after  hardening 
in  85  per  cent,  alcohol  remains  perfectly  transparent.  But  celloidin  or 
common  collodion  also  give  perfectly  transparent  masses  if  cleared  in  bulk 
as  I  recommend  should  be  done  (§§  163 — 165)  ;  so  that  there  is  no  advan- 
tage on  this  head  in  having  recourse  to  photoxylin,  unless  it  be  desired  to 
proceed  in  the  old  way.  Some  writers  say  that  it  gives  a  better  consistency, 
but  others  deny  this  (APATHY,  e.g.). 

152.  Preparation  of  Objects. — The  objects  must  first  be  very 
thoroughly  dehydrated  with  absolute  alcohol.      They  are  then 


122  CHAPTER    IX. 

soaked  till  thoroughly  penetrated  in  ether,,  or,  which  is 
better,  in  a  mixture  of  ether  and  absolute  alcohol.  DUVAL 
(loc.  cit.)  takes  for  this  purpose  a  mixture  of  ten  parts  of  ether 
to  one  of  alcohol ;  SCHIEF^EEDECKEB  (and  the  majority  of 
workers)  a  mixture  of  equal  parts  of  ether  and  alcohol ; 
TUBBY  (in  Nature,  November  17th,  1892,  p.  51)  advises  a 
mixture  of  four  parts  of  ether  and  one  of  alcohol.  But  the 
point  is  one  of  no  great  importance.  FISH  advises  acetone, 
see  next  §. 

This  stage  may  be  omitted  if  the  objects  are  of  a  suffi- 
ciently permeable  nature,  and  they  may  be  brought  direct 
from  alcohol  into  the  collodion  bath. 

153.  The  Collodion  Bath.— The  next  step  is  to  get  the 
objects  infiltrated  with  thick  collodion.  The  secret  of  suc- 
cess here  is  to  infiltrate  them  first  with  thin  solutions,  then 
with  the  definitive  thick  one.  (A  thin  solution  may  be 
taken  to  mean  one  containing  from  4  to  6  per  cent,  of  cel- 
loidiii  [dried  as  described  in  §  151]  ;  a  thick  solution,  one 
containing  10  to  12  per  cent.) 

If  collodion  be  taken,  the  thin  solutions  may  be  made  by 
diluting  it  with  ether.  If  photoxylin  or  cel]oidin  be  taken, 
the  solutions  are  made  in  a  mixture  of  ether  and  absolute 
alcohol  in  equal  parts. 

The  dried  celloidin  shavings  dissolve  very  slowly  in  the 
mixture.  ELSCHNIG  (Zeit.  f.  wi«s.  M>ik  ,  x,  4,  1893,  p.  443) 
states  that  solution  is  obtained  much  quicker  if  the  shavings 
be  first  allowed  to  swell  up  for  twenty-four  hours  in  the 
necessary  quantity  of  absolute  alcohol,  and  the  ether  be 
added  afterwards.  On  trial  it  seems  to  me  that  this  is  so. 

BUSSE  (op.  cit.,  ix,  1,  1892,  p.  47)  gives  the  following  pro- 
portions for  the  successive  baths  : — No.  1,  10  parts  by  weight 
of  photoxylin  or  perfectly  dried  celloidin  to  150  parts  of  the 
ether  and  alcohol  mixture  :  No.  2,  10  parts  of  photoxylin  or 
celloidin  to  105  of  the  mixture  :  No.  3,  10  parts  to  80  of 
the  mixture  (already-used  solution  may  be  employed  for  the 
first  bath). 

I  generally  use  only  two  solutions  :  one  weak  one,  and  one 
strong  one  corresponding  approximately  to  Busse's  No.  2. 
His  No.  3  is  so  thick  that  excessive  time  is  required  to 
obtain  penetration  bv  it. 


COLLODION    AND    OTHER    IMBEDDING    MKTHODS.        123 

FISH  (Journ.  Applied  Microscop.,  ii,  4,  1899,  p.  323)  first  infiltrates 
with  acetone  (which  he  says  may  be  used  as  a  fixing  and  dehydrating 
airent  at  the  same  time),  then  with  a  4  per  cent,  solution  of  pyroxylin  (gun 
cotton)  in  acetone  ;  and,  lastly,  in  an  8  per  cent,  acetone  solution  of  the 
same. 

The  objects  ought  to  remain  in  the  first  bath  until  very 
thoroughly  penetrated; — days,  even  for  small  objects, — 
weeks  or  months  for  large  ones  (human  embryos  of  from 
six  to  twelve  weeks,  for  instance).  If  the  object  contain 
cavities,  these  should  be  opened  to  ensure  their  being  filled 
by  the  mass. 

When  the  object  is  duly  penetrated  by  the  thin  solution, 
or  solutions  if  more  than  one  have  been  employed,  it  should 
be  brought  into  the  thickest  one.  This  may  be  done  (as  first 
described  in  this  work,  1st  edit.,  1885,  p.  194)  by  allowing 
the  thin  sqlution  to  concentrate  slowly  (the  stopper  of  the 
containing  vessel  being  raised,  for  instance  by  means  of  a 
piece  of  paper  placed  under  it) ,  and  making  up  the  loss  from 
evaporation  with  thick  solution. 

APATHY  (Mikrotechnik,  p.  121)  holds,  however,  that  it  is 
preferable  to  transfer  to  fresh  thick  solution,  as  he  finds 
that  a  better  consistency  after  hardening  is  thus  obtained. 

154.  Imbedding. — If  the  object  is  such  that  it  can  be  fixed, 
by  gumming  or  otherwise,  to  the  holder  of  the  microtome 
without  the  intervention  of  any  specially  shaped  mass  of 
collodion  around  it,  and  if  the  presence  of  such  a  mass  be 
not  required  in  the  interest  of  the  orientation  of  the  object, 
or  of  the  production  of  continuous  series  of  sections,  or  of 
very  thin  sections,  no  special  imbedding  is  necessary,  and  as 
soon  as  the  objects  are  duly  penetrated  by  the  thick  solution 
you  may  proceed  to  the  hardening  part  of  the  process.  In 
other  words,  it  is  waste  of  time  to  get  the  object  into  a 
special  block  of  collodion  if  that  is  not  rendered  desirable 
for  the  reasons  above  mentioned.  But  for  fine  and  regular 
work  I  hold  that  it  is  necessary. 

In  that  case  the  objects  must  at  this  stage,  if  it  has  not 
been  done  before,  be  imbedded — that  is,  arranged  in  position 
in  the  thick  collodion  in  the  receptacle  in  which  they  are  to 
be  hardened.  For  the  usual  manipulations  see  §  12(5.  I 
recommend  the  paper  thimbles  or  cylindrical  trays,  Fig.  2, 


124  CHAPTER    IX. 

as  being  very  convenient  for  collodion  imbedding.  The 
bottoms,  however,  should  be  made  of  soft  wood  in  preference 
to  cork ;  cork  is  elastic,  and  bends  in  the  object-holder  of 
the  microtome,  deforming  the  mass  and  object.  The  box 
should  be  prepared  for  the  reception  of  the  object  by  pouring 
into  it  a  drop  of  collodion,  which  is  allowed  to  dry.  The 
object  of  this  is  to  prevent  bubbles  coming  up  through  the 
wood  or  cork  and  lodging  in  the  mass. 

Objects  may  also  be  imbedded  on  a  piece  of  pith  or 
leather,  which  should  also  be  prepared  with  a  layer  of  dry 
collodion. 

Watch  glasses,  deep  porcelain  water-colour  moulds,  and 
the  like,  also  make  convenient  imbedding  receptacles.  Care 
should  be  taken  to  have  them  perfectly  dry. 

It  not  unfrequently  happens  that  during  these  manipula- 
tions bubbles  make  their  appearance  in  the  mass.  Before 
proceeding  with  the  hardening  these  should  be  got  rid  of. 
This  may  be  done  by  exposing  the  whole  for  an  hour  or  two 
to  the  vapour  of  ether  in  a  desiccator  or  other  well-closed 
vessel.  Care  should  be  taken  that  the  ether  (which  may  be 
poured  on  the  bottom  of  the  vessel)  does  not  wet  the  mass 
(BussE,  Zeit.  f.  luiss.  Mik.,  viii,  4,  1892,  p.  467). 

155.  Orientation. — If  it  be  desired  to  mark  the  position  of  the  object 
in  the  mass  in  order  to  facilitate  the  subsequent  orientation  of  it  on  the 
object-holder  of  the  microtome,  recourse  may  be  had  to  the  method  described 
by  ETCLESHYMEE  in  Amer.  Nat.,  xxvi,  1892,  p.  354  (see  also  Journ.  Boy. 
Mic.  Soc.,  1892,  p.  562).  The  object  is  imbedded  in  one  of  the  metal 
boxes  described  in  §  126.  The  box  has  its  ends  and  sides  perforated  at 
regular  intervals  by  small  opposite  holes.  Silk  threads  are  passed  through 
these  holes  from  side  to  side,  stretched,  and  kept  tight  by  sticking  them  to 
the  sides  of  the  box  by  means  of  a  drop  of  celloidin,  leaving  a  length  of  a 
couple  of  inches  hanging  loose.  The  loose  ends  are  soaked  in  thin  celloidin 
solution  with  which  lamp-black  has  been  mixed.  The  object  is  arranged  in 
position  on  the  framework  formed  by  the  taut  threads  in  the  box,  the  mass 
is  poured  in,  and 'the  whole  is  hardened.  After  hardening,  the  celloidin 
holding  the  ends  of  the  threads  is  dissolved  by  means  of  a  drop  of  ether, 
and  the  lampblacked  ends  are  pulled  through  the  box.  This  leaves 
adhering  to  the  bottom  of  the  mass  a  series  of  black  lines  which  form 
orientation  points. 

APATHY  (Zeit.  f.  wits*.  Mik.,  v,  1,  1888,  p.  47)  arranges 
objects  on  a  small  rectangular  plate  of  gelatin,  placed  on  the 
bottom  of  the  imbedding- recipient.  The  gelatin  is  turned 


COLLODION    AND    OTHER    IMBEDDING    METHODS.         125" 

out  with  the  mass  after  hardening,  and  cut  with  it.      The 
c'tlo-es  of  the  gelatin  form  good  orientation  lines. 

HALLE  and  BORN  (see  Zeit.  f.  iviss.  Hik.,  xii,  3,  1896, 
p.  364)  use  plates  of  hardened  white  of  egg,  in  which  a 
shallow  furrow  for  the  reception  of  the  objects  has  been  cut 
by  means  of  a  special  instrument.  See  further  under 
Enibryological  Methods — Reconstruction. 

156.  Hardening,  Preliminary. — This  is  logically  the  next 
step,  but  as  a  matter  of  fact  is  frequently  begun  before. 
For  the  different  processes  of  the  collodion  method  so  run 
into  one  another  that  it  is  difficult  to  assign  natural  lines  of 
demarcation  between  them. 

The  objects  being  imbedded,  and  in  the  stage  at  which  we 
left  them  at  the  end  of  §  155,  the  treatment  should  be  as 
follows  : — The  receptacles  or  supports  are  set  with  the  mass 
under  a  glass  shade  allowing  of  just  enough  communication 
with  the  air  to  set  up  a  slow  evaporation.  Or  porcelain 
moulds  or  small  dishes  may  be  covered  with  a  lightly-fitting 
cover.  As  soon  as  the  added  thick  collodion  (of  which  only 
just  enough  to  cover  the  object  should  have  been  taken)  has 
so  far  sunk  down  that  the  object  begins  to  lie  dry,  fresh 
thick  solution  is  added,  and  the  whole  is  left  as  before.  (If 
the  first  layer  of  collodion  has  become  too  dry,  it  should  be 
moistened  with  a  drop  of  ether  before  adding  the  fresh 
collodion) .  Provision  should  be  again  made  for  slow  evapora- 
tion, either  in  one  of  the  ways  above  indicated,  or,  which  is 
perhaps  better,  by  setting  the  objects  under  an  hermetically 
fitting  bell-jar,  which  is  lifted  for  a  few  seconds  only  once  or 
twice  a  day.  I  have  sometimes  found  it  advantageous  to 
set  the  objects  under  a  bell-jar  together  with  a  dish  con- 
taining alcohol,  so  that  the  evaporation  is  gone  through  in 
an  atmosphere  of  alcohol.  This  is  especially  indicated  for 
very  large  objects.  The  whole  process  of  adding  fresh  col- 
lodion, and  placing  the  objects  under  the  required  conditions 
of  evaporation,  is  repeated  every  few  hours  for,  if  need  be, 
two  or  three  days. 

When  the  mass  has  attained  a  consistency  such  that  the 
ball  of  a  finger  (not  the  nail)  no  longer  leaves  an  impress  on 
it,  it  should  be  scooped  out  of  the  dish  or  mould,  or  have  the 
paper  removed  if  it  has  been  imbedded  in  paper,  and  be 


126  CHAPTER    IX. 

submitted  to  the  next  stage  of  the  hardening  process.  (If 
the  mass  is  found  to  be  not  quite  hard  enough  to  come  away 
safely,  it  should  be  put  for  a  day  or  two  into  weak  alcohol, 
30  to  70  per  cent.) 

157.  Hardening,  Definitive. — Several  methods  are  available 
for  the  definitive  hardening  process.  One  of  these  is  the 
chloroform  method,  due  to  VIALLANKS  (Rech.  sur  I' Hist,  et  le 
Dev.  des  Inwctes,  1883,  p.  129).  I  recommend  this  method 
for  small  objects,  because  I  find  it  much  more  rapid  than  the 
alcohol  method,  whilst  giving  at  least  as  good  a  consistency 
to  the  mass.  For  large  objects  the  method  is  said  by  some 
writers  to  be  inferior  to  the  alcohol  method,  because  the 
rapid  hardening  of  the  external  layers  is  an  obstacle  to  the 
diffusion  necessary  to  the  hardening  of  the  inner  layers. 

The  method  consists  in  bringing  the  objects  into  chloro- 
form. 

Under  the  influence  of  this  reagent  the  collodion  coagu- 
lates rapidly  into  a  mass  having  the  consistency  of  wax. 

In  some  cases  a  few  hours'  immersion  is  sufficient  to  give 
the  requisite  consistence.  In  no  case  have  my  specimens 
required  more  than  three  days.  But  the  length  of  time 
required  varies  in  a  very  inexplicable  way,  so  that  no  rule 
can  be  given.  The  collodion  frequently  becomes  opaque  on 
being  put  into  the  chloroform,  but  regains  ifcs  transparency 
after  a  time. 

Small  objects  may  be  hardened  by  chloroform  'Without  pre* 
liminary  hardening  by  evaporation.  All  that  is  necessary  is 
to  expose  the  mass  to  the  air  for  a  few  seconds  until  a  mem- 
brane has  formed  on  it,  and  then  bring  it  into  chloroform. 
If  the  mass  is  in  a  test-tube  this  may  be  filled  up  with 
chloroform,  and  left  for  two  or  three  days  if  need  be.  By 
this  time  the  collodion  mass  will  be  considerably  hardened, 
and  also  somewhat  shrunk,  so  that  it  can  be  shaken  out  of  ; 
the  tube.  It  is  then  brought  into  fresh  chloroform  in  a 
larger  vessel,  where  it  remains  for  a  few  more  days  until  it 
is  ready  for  cutting.  But  sufficient  hardening  is  sometimes 
obtained  in  a  few  hours. 

Good  chloroform  is  a  necessity,  as  the  reaction  cannot  be 
obtained  with  samples  of  chloroform  that  are  not  free  from 
water. 


COLLODION    AND   OTHElt    IMBEDDING    METHODS.         127 

The  above  processes  are  excellent,  but  I  regard  them  as 
primitive  forms  of  the  chloroform  method.  I  now  almost 
always  harden  in  vapour  of  chloroform.  All  that  is  necessary 
is  to  put  the  liquid  mass  (after  having  removed  bubbles  as 
directed  in  §  154)  with  its  recipient  into  a  desiccator  on  the 
bottom  of  which  a  few  drops  of  chloroform  have  been  poured. 
The  action  is  very  rapid,  and  the  final  consistency  of  the 
mass  at  least  equal  to  that  obtained  by  the  best  alcohol 
hardening.  We  shall  revert  to  this  subject,  §  165. 

The  more  commonly  employed  hardening  method  is  the 
alcohol  method.  The  objects  are  thrown  into  alcohol  and 
left  there  until  they  they  have  attained  the  right  consistency 
(one  day  to  several  weeks) .  The  bottle  or  other  vessel  con- 
taining the  alcohol  ought  not  to  be  iiqhtly  closed,  but  should  be 
left  at  least  partly  open. 

The  strength  of  the  alcohol  is  a  point  on  which  the  prac- 
tice of  different  writers  differs  greatly.  The  question  may 
now  be  considered  to  be  finally  settled  by  experiments 
specially  directed  to  the  clearing  up  of  this  point,  made  by 
BHSSE  (Zeit.  f.  iviss.  Mifo,  ix,  1,  1892,  p.  49),  and  which  I 
have  repeated  and  confirmed.  BUSSE  finds  that  alcohol  of 
about  85  per  cent,  is  the  best,  both  as  regards  the  cutting  con- 
sistency and  the  transparency  of  the  mass.  Care  must  be 
taken  to  keep  masses  hardened  in  this  grade  of  alcohol 
moist  whilst  cutting,  as  they  dry  by  evaporation  very  quickly. 
Some  workers  use  lower  grades,  70  to  80  per  cent.,  or 
even  lower.  APATHY  (Mikrotechnik,  p.  185)  mentions 
"  glycerin-alcohol,"  but  without  giving  details.  BLUM  (Anat. 
Anz.j  xi,  1896,  p.  724)  mentions  "weak  spirit  with  formol 
added  to  it/'  saying  that  formol  hardens  celloidin. 

Lastly,  the  mass  ma)-  be  frozen.  After  preliminary  hardening  by  alcohol 
it  is  soaked  for  a  few  hours  in  water,  in  order  to  get  rid  of  the  greater  part 
of  the  alcohol  (the  alcohol  should  not  be  removed  entirely,  or  the  mass  may 
freeze  too  hard).  It  is  then  dipped  for  a  few  moments  into  gum  mucilage 
in  order  to  make  it  adhere  to  the  freezing  plate,  and  is  frozen.  If  the  mass 
have  frozen  too  hard,  cut  with  a  knife  warmed  with  warm  water. 

A  paper  has  been  written  by  FLOEMAN  (Zeit.  f.  wiss.  Mik.,  vi,  2,  1889, 
p.  184)  to  recommend  that  the  definitive  hardening  should  be  done  without 
the  aid  of  alcohol  or  chloroform,  by  simply  cutting  out  the  blocks,  turning 
them  over,  and  carefully  continuing  the  evaporation  process  in  the  way 
described  above.  I  described  this  process  myself  in  the  first  edition  of 
this  work.  No  doubt  the  author  is -right  in  claiming  for  it  a  superior 


128  CHAPTEB   IX. 

degree  of  hardening  of  the  mass  ;  but  I  doubt  whether  it  is  possible  to  carry 
the  hardening  much  beyond  the  point  attained  by  the  chloroform  or  alcohol 
method  without  incurring  a  very  undesirable  degree  of  shrinkage. 

The  hardening  processes  used  in  the  method  of  clearing 
before  cutting,  which  I  prefer  to  all  the  foregoing,  will  be 
described  later  on,  §  165. 


158.  Preservation. — The  hardened  blocks  of  collodion  may 
be  preserved  till  wanted  in  weak  alcohol  (70  per  cent.). 
They  may  also  be  preserved  dry  by  dipping  them  into  melted 
paraffin  (APATHY,  Zeit.  f.  wiss.  Mik.,  v,  1,  1888,  p.  45),  or, 
after  rinsing  in  water,  in  glycerin-jelly,  which  may  be  re- 
moved with  warm  water  before  cutting  (APATHY,  Mitth.  Zool. 
Stat.  Neapel,  xii,  1897,  p.  372). 

Reference  numbers  may  be  written  with  a  soft  lead  pencil 
on  the  bottom  of  the  paper  trays,  or  with  a  yellow  oil  pencil 
on  the  bottom  of  the  watch  glasses  in  which  the  objects  are 
imbedded.  On  removal  of  the  paper  from  the  collodion 
after  hardening,  the  numbers  will  be  found  impressed  on  the 
collodion. 

159.  Cutting. — If  the  object  has  not  been  stained  before  imbedding,  it 
may  form  so  transparent  a  mass  with  the  collodion  that  the  arrangement  of 
the  object  and  sections  in  the  right  position  may  be  rendered  very  difficult. 
It  is,  therefore,  well  to  stain  the  collodion  lightly,  just  enough  to  make  its 
outlines  visible  in  the  sections.  This  may  be  done  by  adding  picric  acid  or 
other  suitable  colouring  matter  dissolved  in  alcohol  to  the  collodion  used  for 
imbedding,  or  to  the  oil  used  for  clearing. 

To  fix  a  collodion  block  to  the  microtome  proceed  as 
follows.  Take  a  piece  of  soft  wood,  or,  for  very  small 
objects,  pith,  of  a  size  and  shape  adapted  to  fit  the  holder 
of  the  microtomy.  Cover  it  with  a  layer  of  collodion,  which 
you  allow  to  dry.  Take  the  block  of  collodion  or  the  in- 
filtrated and  hardened  but  not  imbedded  object,  and  cut  a 
slice  off  the  bottom,  so  as  to  get  a  clean  surface.  Wet  this 
surface  first  with  absolute  alcohol,  then  with  ether  (or  allow 
it  to  dry);  place  one  drop  of  very  thick  collodion  on  the  pre- 
pared wood  or  pith  and  press  down  tightly  on  to  it  the  wetted 
or  dried  surface  of  the  block  or  object.  Then  throw  the 
whole  into  weak  (70  per  cent.)  alcohol  for  a  few  hours,  or 


COLLODION    AND    OTHER    IMBEDDING    METHODS.        129 

even  less,  or  better  into  chloroform,  or  vapour  of  chloroform, 
for  a  few  minutes,  in  order  that  the  joint  may  harden 

Dr.  LINDSAY  JOHNSON  informs  me  that  he  finds  it  very 
convenient  to  take  for  this  purpose  the  cement  used  by  metal 
turners  for  fastening  metal  objects  on  to  boxwood  chucks. 
The  exact  composition  of  this  cement  varies  somewhat,  but 
an  average  one  is — beeswax,  1  part ;  rosin,  2  parts.  To  use 
it  you  must  get  the  block  of  celloidin  perfectly  dry  at  the 
bottom,  then  warm  the  object-holder  slightly,  if  possible 
over  a  flame ;  drop  on  to  it  a  few  drops  of  melted  cement, 
and  press  on  to  it  the  block  of  collodion,  which  will  be  firmly 
fixed  as  soon  as  the  cement  is  cool — that  is  in  a  few  seconds. 

For  objects  of  any  considerable  size  it  is  important  not  to 
use  cork  for  mounting  on  the  microtome,  especially  if  the 
object-holder  be  a  vice  ;  for  cork  bends  under  the  pressure  of 
the  holder,  and  the  elastic  collodion  bends  with  it,  deforming 
the  object.  I  have  seen  large  embryos  so  deformed  in  this 
way  that  the  sections  obtained  were  true  calottes,  segments 
of  a  sphere.  If  the  object-holder  be  of  the  cylinder  type, 
as  in  the  later  forms  of  the  Thoma  microtome,  the  above - 
described  accidents  will  be  less  likely  to  happen,  and  a  good 
cork  may  be  used ;  but  even  then,  I  think,  wood  is  safer. 
G-AGE  has  recommended  bits  of  glass  cylinders.  JELINEK 
(Zeii.  f.  wiss.  Mil:.,  xi,  2,  1894,  p.  237)  recommends  a  sort  of 
vulcanite  known  as  "  Stabilit,"  which  is  manufactured  for 
electrical  insulation  purposes.  It  is  supplied  in  suitable 
blocks  by  JUNG  (presumably  also  obtainable  through  GRUBLER 
AND  Co.). 

Sections  (from  such  masses  as  have  not  been  cleared 
before  cutting)  are  cut  with  a  knife  kept  abundantly  wetted 
with  alcohol  (of  50  to  85  or  even  95  per  cent.).  Some  kind 
of  drip  arrangement  will  be  found  very  useful  here.  Apathy 
recommends  that  the  knife  be  smeared  with  yellow  vaselin  ; 
it  cuts  better,  is  protected  from  the  alcohol,  and  the  mobility 
of  the  alcohol  on  the  blade  is  lessened. 

The  knife  is  set  in  as  oblique  a  position  as  possible. 

Very  brittle  sections  may  be  collodionised  as  explained 
above  (§  136). 

The  sections  are  either  brought  into  alcohol  (of  50  to  85 
or  95  per  cent.)  as  fast  as  they  are  made ;  or  if  it  be  desired 
to  mount  them  in  series,  they  are  treated  according  to  one  of 

9 


130  CHAPTER    IX. 

the  methods   described   below,   in   the    chapter   on    "  Serial 
Section  Mounting." 

Masses  that  have  been  cleared  before  cutting  with  cedar 
oil,  or  the  like,  may  be  cut  dry,  §  165. 

160.  Staining. — The  sections  may  now  be  stained  as  desired* 
either  loose,  or  mounted  in  series  on  slides  or  on  paper  as 
described  in  the  chapter  on  "  Serial  Section  Mounting."      It 
is  not  in  general  necessary,  nor  indeed  desirable,  to  remove 
the  mass  before  staining,  as  it  usually  either  remains  colour- 
less, or  gives  up  the  stain  on  treatment  with  alcohol.      But 
some  of  the  anilin  dyes  and  some  other  colours  stain  it  strongly, 
.and  are  not  removed  with  sufficient  completeness  by  the  pro- 
cesses   of    dehydration   and  clearing.      If    it    be    desired    to 
employ  these,   the   mass   may  be   removed  by  treating  the 
sections  with  absolute  alcohol  or  ether. 

161.  Clearing  and  Mounting. — You  may  mount  in  glycerin 
without  removing  the  mass,  which  remains  as  clear  as  glass 
in  that  medium. 

You  may  mount  in  balsam,  also,  without  removing  the 
mass,  which  does  no  harm,  and  serves  the  useful  purpose  of 
holding  the  parts  of  the  sections  together  during  the 
manipulations.  Dehydrate  in  alcohol  of  95  or  96  per  cent, 
(not  absolute,  as  this  attacks  the  collodion).  NIKIFOEOW 
(Zeit.  f.  wiss.  Mik.,  viii,  2,  1891,  p.  189)  recommends  a  mix- 
ture of  equal  parts  of  alcohol  and  chloroform.  Clear  with 
a  substance  that  does  not  dissolve  collodion.  The  clearing 
agents  most  recommended  are  origanum  oil  (01.  Origan. 
Cretici,  it  is  said,  should  be  taken,  not  01.  Orig.  Gallici ; 
but  see  as  to  this  reagent  the  remarks  in  Chap.  VI,  §  113), 
bergamot  oil  (said  to  make  sections  shrink  somewhat),  oil  of 
sandal-wood,  lavender  oil,  oil  of  cedar-wood  (safe  and  gives 
excellent  results,  but  acts  rather  slowly),  chloroform,  xylol,  or 
benzol  (may  make  sections  shrink  if  not  well  dehydrated),  or 
Dunham's  mixture  of  three  or  four  parts  of  white  oil  of 
thyme  with  one  part  of  oil  of  cloves.  (As  to  oil  of  thyme, 
see  also  "  Origanum  Oil"  in  Chap.  VI,  §  113.) 

FISH  (Proc  Amer.  Mic.  Soc.,  1893)  advises  a  mixture  of 
one  part  of  red  oil  of  thyme  with  three  parts  of  castor  oil, 
the  latter  being  added  in  order  to  counteract  the  volatility 


COLLODION   AND    OTHER    IMBEDDING    METHODS.         131 

of  the  thyme  oil.  But  later  (June,  1895),  writing  to  me, 
Dr.  Fish  says  he  has  substituted  the  white  oil  of  thyme  for 
the  red,  and  finds  it  an  advantage  in  orientating.  See  also 


Some  specimens  of  clove  oil  dissolve  collodion  very  slowly,  and  may  be 
used,  but  I  would  not  be  understood  to  recommend  it.  The  action  of 
origanum  oil  varies  much,  according  to  the  samples  ;  some  sorts  do  not 
clear  the  collodion,  others  dissolve  it,  others  pucker  it.  MIXOT  (Zeit.  f.  wiss. 
3/Vfc.,  iii,  2,  1886,  p.  175)  says  that  Dunham's  mixture  "clarifies  the  sec- 
tions very  readily,  and  softens  the  celloidin  just  enough  to  prevent  the 
puckering,  which  is  so  annoying  with  thyme  alone." 

Carbolic  acid  has  been  recommended.  WEIGEET  (Zeit.  f.  wiss.  Mik.,  iii, 
4,  1866,  p.  480)  finds  that  a  mixture  of  3  parts  of  xylol  with  1  part  of 
carbolic  acid  (anhydrous)  clears  well.  But  it  must  not  be  used  with  the 
basic  anilin  stains,  as  it  discolours  them.  For  these  anilin  oil  may  be  used 
with  the  xylol  in  the  place  of  carbolic  acid. 

Anilin  oil  clears  well  (it  will  clear  from  70  per  cent,  alcohol),  but  unless 
thoroughly  removed  the  preparation  becomes  yellowish-brown.  This  colora- 
tion may  be  removed  by  soaking  in  chloroform  for  twenty-four  hours  (see 
VAN  GIESON,  Amer.  Mon.  Mic.  Joum.,  1887,  p.  49,  or  Journ.  Roy.  Mic. 
Soc.,  1887,  p.  519,  for  a  review  of  these  clearing  agents  ;  see  also  §  121). 

Beech-wood  creasote  has  been  recommended  (by  M.  Flesch). 

EYCLESHYMER  (Amer.  Nat.,  xxvi,  1892,  p.  354  ;  Journ.  Roy.  Mic.  Soc., 
1892,  p.  565)  advises  a  mixture  of  equal  parts  of  bergamot  oil,  cedar  oil,  and 
carbolic  acid. 

For  oil  of  cajeput  see  §  116;  and  for  this  and  other 
clearers  see  also  the  paper  of  JOBDAN  quoted  §  107. 

162.  Review  of  the  Older  Celloidin  Method.  —  The  older  cel- 
loidin method,  described  in  the  foregoing  pages,  is  extremely 
lengthy  and  cumbrous.  The  operation  of  infiltrating  the 
tissues  with  the  collodion  requires  days  or  weeks.  The 
hardening  process  frequently  requires  nearly  as  much  time. 
The  resulting  mass  has  the  disadvantage  of  being  opaque,  or 
at  most  only  translucent,  not  transparent.  The  mass  has  to 
be  cut  under  the  surface  of  alcohol,  or  at  least  with  constant 
wetting  with  alcohol,  and  with  a  knife  kept  constantly  wet 
with  alcohol.  By  the  recent  method  of  clearing  the  mass 
before  cutting  a  large  part  of  these  defects  is  done  away 
with  ;  the  resultant  mass  is  as  clear  as  glass,  thus  allowing  the 
most  perfect  orientation  of  the  object  ;  and,  as  I  have  shown 
(LEE  et  HKNNEGOY,  Traite  des  Mtthodes  techniques  de  I'Anat. 
mic.,  1896,  p.  230),  the  mass  can  with  advantage  be  cut  dry, 


132  CHAPTER    IX. 

thereby  greatly  simplifying  the  operation  of  cutting.  By 
GILSON'S  ingenious  Rapid  Method,  the  time  necessary  for 
hardening  is  very  greatly  abridged,  and  the  whole  series 
of  operations  becomes  almost  as  short  and  simple  as  the 
paraffin  method.  I  cannot  imagine  that  anyone  who  has 
ever  employed  the  new  method  would  willingly  go  back  to 
the  old  one.  The  following  paragraphs  describe  the  new 
method. 

163.  The   New  Method,  by  Clearing   before    Cutting.— This 
process  is  due,  I  believe,  in  the  first   instance  to   E.  MEYER 
(Biol.  Centralb.,  x,  1890,  p.  508),  who  advised  soaking  blocks 
before  cutting  for  twenty-four  hours   in   glycerin.      Bo  MFCS 
(Amer.  Anat.,  xxvi,  1892,  p.  80  ;   see  Jonrn.  Roy.  Mic.  Soc., 
1892,  p.  438)  advises  clearing  the  mass,  after  hardening  in 
chloroform,  with  white  oil   of  thyme  or  other  suitable  clear- 
ing agent  (see  above,  §  161).      The  knife  is  wetted  with  the 
clearing  oil,  and  the  same  oil  is  employed  for  covering  the 
exposed  surface  of  the  object  after  each  cut.      Similar  recom- 
mendations  are   made   by   EYCLESHYMKI*    (op.  cit.,   pp.   354, 
563),  carbolic  acid,  or  glycerin,  or  the  mixture  given  §  161, 
being  suggested  for  clearing ;  and  Professor  GTILSON  has  for 
a  long  time   past   adopted   the   practice  of   clearing  before 
cutting  with  cedar  oil,  as  described  in  the  next  §. 

FISH  (loc.  cit.,  §  161)  also  advocates  the  practice  of  clear- 
ing in  the  mass,  recommending  the  clearing  mixture  there 
given.  Similarly  G-AGE,  Trans.  Amer.  Mic.  Soc.,  xvii,  1896, 
p.  361. 

All  the  authors  above  quoted  cut  in  the  wet  way,  that  is 
to  say,  with  a  knife  wetted  with  the  clearing  liquid.  I  have 
found  a  great  improvement  in  cutting  dry,  and  in  employing 
the  combined  hardening  and  clearing  process  of  GriLsoisr, 
given  below. 

164.  GTILSON'S  Rapid  Process    (communicated  by   Professor 
G-ILSON,  April,  1892). — The  object  is   dehydrated,  soaked  in 
ether,    and  brought  into  a  test-tube  with  collodion  or  thin 
celloidin  solution.      The  tube  is  dipped  into  a  bath  of  melted 
paraffin,  and  the  collodion  allowed  to  boil  (which  it  does  at 
a  very  low  temperature)  until  it  has  become  of  a  syrupy  con- 
sistence.    (It  should  be  boiled  down  to  about  one  third  of  its 


COLLODION    AND    OTHER   IMBEDDING    METHODS.         133 

volume.)  The  mass  is  then  turned  out,  mounted  on  a  block 
of  hardened  celloidin,  and  the  whole  hardened  in  chloroform 
or  in  a  mixture  of  chloroform  and  cedar  oil  for  about  an  hour. 
It  is  then  cleared  in  cedar  oil  (if  hardened  in  pure  chloro- 
form :  special  clearing  will  not  be  necessary  if  it  has  been 
hardened  in  the  mixture).  It  may  now  be  fixed  in  the 
microtome  and  cut,  using  cedar  oil  to  wet  the  knife,  and 
cover  the  exposed  surface  of  the  object  after  each  cut. 

It  will  be  observed  that  this  process  is  very  much  more 
rapid  than  the  old  process  in  two  ways.  The  celloidin 
bath,  being  given  warm,  is  greatly  abridged ;  small  objects 
can  be  duly  infiltrated  in  an  hour,  where  days  would  be 
required  by  the  old  process.  And  the  hardening  is  very 
much  more  rapid  than  hardening  by  alcohol,  which  requires 
at  least  twenty-four  hours.  As  collodion  boils  at  a  very  low 
temperature  very  little  heat  is  required,  and  there  is  no  risk 
of  the  tissues  suffering  on  that  head. 

165.  The  Dry  Cutting  Method. — I  recommend  the  following 
as  being  a  further  improvement.  Infiltrate  with  collodion 
or  celloidin  either  by  GILSON'S  process,  or  by  soaking  in  the 
cold  in  the  usual  way,  §  153.  This  is  a  much  slower  pro- 
cess, but  does  not  take  up  more  of  the  worker's  time,  as  the 
specimens  require  no  attention  whilst  in  the  bath.  Imbed 
as  usual,  either  directly  on  the  holder  of  the  microtome,  or 
in  a  paper  tray  or  a  water-colour  mould  or  the  like.  Harden 
in  vapour  of  chloroform  for  from  one  hour  (generally  suf- 
ficient for  small  objects)  to  overnight.  This  is  done  by 
putting  the  object  (definitively  imbedded  in  the  final  thick 
solution,  but  without  any  preliminary  hardening  in  the  air) 
into  a  Steinach's  sieve-dish  or  into  a  desiccator,  on  the 
bottom  of  which  a  teaspoonful  of  chloroform  has  been 
poured.  (The  objects  may  remain  for  months  in  the  chloro- 
form vapour  if  desired.)  As  soon  as  the  mass  has  attained 
sufficient  superficial  hardness,  it  is,  of  course,  well  to  turn  it 
out  of  its  recipient,  and  turn  it  over  from  time  to  time,  in 
order  that  it  may  be  equally  exposed  on  all  sides  to  the 
action  of  the  vapour.  When  fairly  hard  (it  is  not  necessary 
to  wait  till  the  mass  has  attained  all  the  hardness  of  which 
it  is  susceptible)  throw  it  into  GILSON'S  mixture.  This 
should  be  at  first  a  mixture  of  one  part  of  chloroform  with 


134  CHAPTER   IX. 

one  or  two  parts  of  cedar  oil.  From  time  to  time  more 
cedar  oil  should  be  added,  so  as  to  bring  the  mixture  up 
gradually  to  nearly  pure  cedar  oil.  As  soon  as  the  object 
is  cleared  throughout,  the  mass  may  be  exposed  to  the  air,, 
and  the  rest  of  the  chloroform  will  evaporate  gradually. 
The  block  may  now  be  mounted  on  the  holder  of  the  micro- 
tome with  a  drop  of  thick  collodion,  §  159,  and  may  either 
be  cut  at  once,  or  may  be  preserved  indefinitely  without 
change  in  a  stoppered  bottle.  Cut  dry,  the  cut  surface  will 
not  dry  injuriously  under  several  hours.  The  cutting  quality 
of  the  mass  is  often  improved  by  allowing  it  to  evaporate  in 
the  air  for  some  hours. 

The  hardening  may  be  done  at  once  in  the  chloroform  and 
cedar- wood  mixture,  instead  of  the  chloroform  vapour,  but  I 
find  the  latter  process  preferable,  as  giving  a  better  harden- 
ing. And  clearing  may  be  done  in  pure  cedar  oil  instead 
of  the  mixture,  but  then  it  will  be  very  slow,  whereas  in  the 
mixture  it  is  extremely  rapid. 

166.  Double  Imbedding  in  Collodion  and  Paraffin. — This  com- 
plicated process  is  sometimes,  though  rarely,  employed  for  objects  of  which 
it  is  desired  to  have  very  thin  sections,  and  which  are  too  brittle  to  give 
good  sections  by  the  plain  paraffin  process.  I  do  not  think  that  in  any  form, 
hitherto  published  it  can  be  considered  to  be  a  success. 

KULTSCHITZKY'S  Method  (Zeit.f.  wiss.  Mik.,  iv,  1,  1887,  p.  48).— After 
the  collodion  bath,  the  object  is  soaked  in  oil  of  origanum  (Oleum  Origani 
vulg.).  It  is  then  brought  into  a  mixture  of  origanum  oil  and  paraffin 
heated  to  not  more  than  40°  C.,  and  lastly  into  a  bath  of  pure  paraffin. 

The  mass  may  be  preserved  in  the  dry  state,  and  may  be  cut  dry. 

RYDEE  (Queen's  Micr.  Bull,  1887,  p.  43  ;  Journ.  Roy.  Mic.  Soc.,  1888, 
p.  512)  modified  the  process  by  substituting  chloroform  for  the  origanum  oil. 

IDE  (La  Cellule,  vii,  1891,  p.  347,  and  viii,  1,  1892,  p.  114)  employed 
with  success  the  following  method  :  —The  object  is  imbedded  in  collodion  in 
a  tube  by  GILSON'S  process  (supra,  §  164)  ;  the  collodion  is  boiled  for  forty 
minutes,  then  brought  for  fifteen  minutes  (this  is  for  small  objects)  into 
chloroform  heated  to  30°  C.  containing  one  fourth  part  of  paraffin  dissolved 
in  it,  then  for  ten  minutes  into  pure  melted  paraffin. 

FIELD  and  MAETIN  (Bull  Soc.  Zool.de  France,  1894,  p.  48),  finding  that 
it  is  difficult  to  get  hardened  celloidin  masses  adequately  impregnated  with 
the  paraffin,  have  worked  out  the  following  process  of  simultaneous 
imbedding.  A  solution  of  dried  celloidin  in  a  mixture  of  equal  parts  of 
absolute  alcohol  and  toluene,  of  about  the  consistency  of  clove  oil,  is  made. 
This  solution  is  saturated  with  paraffin,  added  in  shavings  at  a  temperature 
not  exceeding  20°  to  23°  C.  The  tissues  are  prepared  by  soaking  in  some  of 
the  mixture  of  alcohol  and  toluene,  and  are  then  penetrated  with  the 


COLLODION    AND   OTHER   IMBEDDING   METHODS.         135 

celloidin-paraffin  solution.  The  mass  is  hardened  by  throwing  it  into  a 
saturated  solution  of  paraffin  in  chloroform  or  in  toluene,  and  is  finally 
imbedded  in  pure  paraffin  in  the  usual  way. 

See  also  the  modifications  of  DAHLGEEN,  Journ.  Applied  Microsc.,  1898,. 
p.  ^7  (Journ.  Roy.  Mic.  Soc.,  1898,  p.  489) ;  SABUSSOW,  Mitth.  Zool  Stat. 
Neapel,  xii,  1896,  p.  353  ;  MITEOPHANOW,  Arch.  Zool.  Exper.  [3],  3,  1896,. 
p.  617). 

Other  Cold  Masses. 

167.  Joliet's  Gum  and  Glycerin  Method  (Arch.  Zool.  Exper. 
et  Gen.,  x,  1882,  p.  xliii ;  Journ.  Roy.  Mic.  Soc.  [N.S.],  iir 
1882,  p.  890). — Pure  gum  arable  dissolved  in  water  to  the 
consistency  of  a  thick  syrup.  (Solutions  of  gum  sold  under 
the  name  of  strong  white  liquid  glue  ["  colle  forte  blanche 
liquids  a  froid  "]  may  also  be  used  ;  they  have  the  advantage 
of  having  a  uniform  consistency.*)  Pour  a  little  of  the 
solution  into  a  watch  glass,  so  as  not  quite  to  fill  it,  add 
from  6  to  10  drops  of  pure  glycerin,  stir  until  thoroughly 
mixed.  In  the  winter  or  in  rainy  weather  less  glycerin 
should  be  taken  than  in  the  summer  or  dry  weather. 

The  object  is  imbedded  in  the  mass  in  the  watch  glass, 
and  the  whole  left  to  dry  for  from  one  to  four  days.  When 
it  has  assumed  a  cartilaginous  consistency,  a  block  contain- 
ing the  object  is  cut  out,  turned  over,  and  allowed  to  dry 
again  until  wanted  for  use.  A  stove,  or  the  sun,  may  be 
employed  for  drying,  but  it  is  best  to  dry  slowly  at  the 
normal  temperature. 

This  process  may  render  service  occasionally  in  the  study 
of  extremely  watery  organisms,  such  as  Salpa,  or  the  Cteno- 
phora. 

168.  STEICKEE'S  Gum  Method  (Hdb.  d.  Gewebel.,  p.  xxiv). — A  concen- 
trated solution  of  gum  arabic.     The  object  is  imbedded  in  the  gum  in  a 
paper  case.     The  whole  is  thrown  into  alcohol,  and  after  two  or  three  days 
may  be  cut.     The  alcohol  should  be  of  about  80  per  cent.  (MATES). 

I  have  seen  masses  of  sufficiently  good  consistency  prepared  by  this  simple 
method. 

169.  ROBEBTSON'S  Grape-sugar   Method,  see  Journ.  of  Anat.  and, 
Physiol.,  xxiv,  1890,  p.  230;  Zeit.f.  wiss.  Mik.,  vii,  1,  1890,  p.  33. 

*  It  is  highly  probable  that  these  commercial  preparations  contain  gelatin, 
and  perhaps  some  other  gum  besides  gum  arabic. 


136  CHAPTER    IX. 

170.  HYATT'S  Shellac  Method,  see  Am.  M.  Mic.  Journ.,  i,  1880,  p.  8  ; 
Journ.  Boy.  Mic.  Soc.,  iii,  1880,  p.  320. 

This  process  is  merely  intended  for  the  purpose  of  making  sections  through 
hard  chitinous  organs  consisting  of  several  pieces,  such  as  stings  and  ovi- 
positors, retaining  all  the  parts  in  their  natural  positions. 

171.  BKUNOTTI'S  Cold  Gelatin  Mass  has  been  given  §  148. 


Masses  for    Grinding   Sections.* 

172.  G.  VON  KOCH'S  Copal  Method  (Zool.  Anz.,  2,  vol.  i,  1878, 
p.  36).^Small  pieces  of  the  object  are  stained  in  bulk  and 
dehydrated  with  alcohol.  A  thin  solution  of  copal  in  chloro- 
form is  prepared  by  triturating  small  fragments  of  copal  in  a 
mortar  with  fine  sand,  pouring  on  chloroform  to  the  powder 
thus  obtained,  and  filtering.  The  objects  are  brought  into 
a  capsule  filled  with  the  copal  solution.  The  solution  is  now 
slowly  evaporated  by  gently  heating  the  capsule  on  a  tile  by 
means  of  a  common  nig*ht-light  placed  beneath  it.  As  soon 
as  the  solution  is  so  far  concentrated  as  to  draw  out  into 
threads  that  are  brittle  after  cooling,  the  objects  are  removed 
from  the  capsule  and  placed  to  dry  for  a  few  days  on  the  tile, 
in  order  that  they  may  more  quickly  become  hard.  When 
they  have  attained  such  a  degree  of  hardness  that  they  cannot 
be  indented  by  a  finger-nHil,  sections  are  cut  from  them  by 
means  of  a  fine  saw.  The  sections  are  rubbed  down  even 
and  smooth  on  one  side  with  a  hone,  and  cemented,  with  this 
side  downwards,  to  a  slide,  by  means  either  of  Canada  balsam 
or  copal  solution.  The  slide  is  put  away  for  a  few  days  more 
on  the  warmed  tile.  As  soon  as  the  cement  is  perfectly  hard 
the  sections  are  rubbed  down  on  a  grindstone,  and  then  on  a 
hone,  to  the  requisite  thinness  and  polish,  washed  with  water, 
and  mounted  in  balsam. 

The  process  may  be  varied  by  imbedding  the  objects 
unstained,  removing  the  copal  from  the  sections  by  soaking 
in  chloroform,  decalcifying  them  if  necessary,  and  then 
staining. 

It  is  sometimes  a  good  plan,  after  removing  the  copal,  to 
cement  a  section  to  a  slide  by  means  of  hard  Canada  balsam, 

*  For  the  manipulations  of  section-grinding,  see  the  Treatises  on  the 
Microscope,  particularly  CARPENTER'S,  The  Microscope  and  Us  Revelations 
(J.  and  A.  Churchill,  London). 


COLLODION   AND    OTHEE    IMBEDDING    METHODS.        137 

then  decalcify  cautiously  the  exposed  half  of  the  specimen, 
wash,  and  stain  it.  In  this  way  von  Koch  was  able  to 
demonstrate  the  most  delicate  lamellae  of  connective  tissue 
in  Isis  elongata. 

This  method  was  imagined  in  order  to  enable  the  hard  and 
soft  parts  of  corals  to  be  studied  in  their  natural  relations. 
It  is  evidently  applicable  to  the  study  of  any  structures  in 
which  hard  and  soft  parts  are  intimately  combined.  For 
purposes  such  as  these  it  is  certainly  a  method  of  the  greatest 
value. 

173.  EHRKNBAUM'S  Colophonium  and  Wax  Method    (Zeit.  f. 
pits.  J//fc.,  1884,  p.  414). — Ehrenbaum  recommends  that  the 
objects  be  infiltrated  with  a  mass  consisting  of  ten  parts  of 
colophonium  to  one  of  wax.      The  addition  of  wax  makes  the 
mass  less  brittle.      Sections  are  obtained  by  grinding  in  the 
usual  way.      The  mass  is  removed  from  them  by  means  of 
turpentine  followed  by  chloroform. 

174.  JOHNSTONE-LAVIS  and  VOSMAER'S  Balsam  Method  (Journ. 
Roy.  Hie.  Soc.,  1887,  p.  200)  — Alcohol  material  is  carefully 
and  gradually  saturated,  first  with  benzol,  and  then  with  thin 
and  thick  solution  of  benzol-balsam.      It  is  then  dried  for  a 
day  in  the  air  and  for  several   days   more  in  a  hot-air  bath. 
When  hard   it  is   ground    in  the    usual    way.      For  further 
details  and  figure  of  the  dry  ing- stove  see  the  original,  which 
claims  for  the  method  several  advantages  over  that    of  von 
Koch. 

175.  WEIL'S  Canada  Balsam  Method  (Zeii.f.  iciss.  Mik  ,  v,  2,  1888, 
p.  L'lH)  ;  Journ.  Roy.  Mic.  Soc.,  1888,  p.  1042). 

176.  GIESBEECHT'S  Shellac  Method.— For  hard  parts  only,  spines  of 
Echinus,  shell,  etc.,  see  Morph.  Jahrb.,  vi,  1880,  p.  95,  or  the  abstract  in 
LEE  und  MAYEB,  Grundziige,  etc. 

Congelation   Masses. 

177.  The  Freezing  Method. — Fresh  tissues  may  be,  and  are, 
frequently  frozen  without  being  included  in  any  mass,  and  in 
certain  cases  very  satisfactory  sections  can  be  obtained  in  this 
manner.  But  the  formation  of  ice  crystals  frequently  causes 


138  CHAPTER    IX. 

tearing  of  delicate  elements,  and  it  is  better  to  infiltrate  the 
tissues  with  a  mass  that  does  not  crystallise  in  the  freezing 
mixture,  but  becomes  hard  and  tough.  HAMILTON  (Journ. 
of  Anat.  and  Phys.,  xii,  1878,  p.  254)  soaked  tissues  in 
syrup  of  a  particular  strength,  viz.  double  refined  sugar,  2 
ounces ;  water,  1  fluid  ounce ;  then  washed  the  superfluous 
syrup  from  the  surface,  and  put  into  ordinary  gum  mucilage 
for  an  hour  or  so,  and  then  imbedded  in  the  freezing  micro- 
tome with  mucilage  in  the  usual  way. 

178.  Gum  and   Syrup    Congelation  Mass  (COLE,  Methods  of 
Microscopical  Research,  1884,  p.  xxxix  ;  Journ.  Boy.  Mic.  Soc. 
[N.S.],  iv,  1884,  p.  318).— Gum  mucilage   (B.P.),  5  parts; 
syrup,  3  parts.      (For  brain  and  spinal  cord,  retina,  and  all 
tissues  liable  to  come  in  pieces  put  4  parts  of  syrup  to  five 
of  gum).      Add  5  grains  of  pure  carbolic  acid  to  each  ounce 
of  the  medium. 

(Gum  mucilage  [B.P.]  is  made  by  dissolving  4  ounces  of 
picked  gum  acacia  in  6  ounces  of  water. 

The  syrup  is  made  by  dissolving  1  pound  of  loaf  sugar  in 
1  pint  of  water  and  boiling.) 

This  medium  is  employed  for  soaking  tissues  previous  to 
freezing. 

The  freezing  is  conducted  as  follows  : — The  gum  and  syrup 
is  removed  from  the  outside  of  the  object  by  means  of  a  cloth  ; 
the  spray  is  set  going  and  a  little  gum  mucilage  painted  on 
the  freezing  plate  ;  the  object  is  placed  on  this  and  surrounded 
with  gum  mucilage  ;  it  is  thus  saturated  with  gum  arid  syrup, 
but  surrounded  when  being  frozen  with  mucilage  only.  This 
combination  prevents  the  sections  from  curling  up  on  the 
one  hand,  or  splintering  from  being  too  hard  frozen  on  the 
other.  Should  freezing  have  been  carried  too  far,  wait  for 
a  few  seconds. 

179.  Dextrin    Congelation  Mass  (WEBB,  The  Microscope,  ix, 
1890,  p.  344;  Journ.   Roy.  Mic.   Soc.,  1890,  p.  113).— Thick 
solution    of    dextrin    in   solution   of  carbolic    acid   in    water 
(1  in  40). 

180.  Gelatin  (SOLLAS,  Quart.  Journ.  Mic.  Soc.,  xxiv,  1884,  pp.  163, 
164;  Journ.  Roy.  Mic.  Soc.  [N.S.],  iv,  1884,  p.  316).  Gum  Gelatin 
(JACOBS,  Amer.  Natural.,  1885,  p.  734 ;  Journ.  Roy.  Mic.  Soc.,  1885, 


COLLODION    AND    OTHER    IMBEDDING   METHODS.        139 

p.  900).  White  of  Egg  (EoLLETT,  Denkschr.  math,  naturw.  Kl.  k.  Acad. 
Wiss.  Wien,  1885  ;  Zeit.  f.  wiss.  Mik.,  1886,  p.  92).— Small  portions  of  tissue 
brought  in  the  white  of  a  freshly  laid  egg  on  to  the  freezing  stage,  frozen, 
and  cut.  Oil  of  Aniseed  (KiJHNE,  Centralb.  f.  Bakteriol.,  xii,  1892, 
p.  28 ;  Journ.  Roy.  Mic.  Soc.,  1892,  p.  706 ;  V.  A.  MOOBE,  Amer.  Mon. 
Mic.  Journ.,  1894,  p.  373  ;  Journ.  Roy.  Mic.  Soc.,  1895,  p.  247). 

For  details  of  these  see  previous  editions. 

For  DOLLKEN'S  method  of  solidifying  Formol  by  means  of  Resorcin,  see 
Zeit.  f.  wiss.  Mik.,  xiv,  1,  1897,  p.  33 ;  Journ.  Roy.  Mic.  Soc.,  1897, 
p.  448. 


CHAPTER  X. 

SERIAL   SECTION    MOUNTING. 

181.  Choice  of  a  Method. — I  recommend  for  general  work 
the  following  : — For  paraffin  sections  that  do  not  require  to 
be  flattened  or  stained,  Schallibaunr's  collodion.      For  paraf- 
fin sections  that  are  to  be  stained  on  the  slide,   the  water 
method,    or    Mayer's    albumen.       For     collodion     sections, 
Mayers  albumen.       For  very  large  collodion  sections,  Wei- 
gert's  process. 

Methods  for  Paraffin  Sections. 

182.  The  Water    Method. —  GAULE  (Arch.  f.  Anat.  u.  Phys., 
Phys.  Abth.,  1881,  p.    156);   SUCHANNEK  (Zeit.  f.  iviss.  Mik., 
vii,   4,    1891,   p.    464)  ;  GDLLAND    (Journ.   Anat.   and  Phys., 
xxvi,  1891,  p.  56)  ;  SCHJEFFERDECKER  (Zeit.  f.  wiss.  Mik.,  ix, 
2,    1892,   p.   202)  ;   HEIDENHAIN    (Kern  und   Protoplasma,  p. 
114)  ;   NUSBAUM  (Anat.  Anz.,  xii,  2,  1896,  p.  52)  ;  MAYER  in 
the  Grundzuge,  LEE  und  MAYER,   1898,  p.    113;  De    GROOT 
(Zeit.  f.  wiss.  Mik.,  xv,  1,  1898,  p.  62),  and  others  (some  ir- 
rational variations  have  been    suppressed). — The    principle 
of  this  method  is  that  the  sections  are  made  to  adhere  to  the 
slide  by    the  mere  molecular  adhesion  of  intimate    contact, 
without  the  intervention   of  any   cementing   substance  ;  the 
sections  being  brought  into  this  intimate   contact  by  being 
slowly  drawn  down  by  the  evaporation   of  a  layer  of  water 
on  which  they  are  floated.      It  is  now  practised,  with  un- 
essential variations,  as  follows  : 

(a]  For  sections  that  are  large  and  not  numerous.  The 
sections  are  flattened  out  on  water  by  one  or  other  of  the 
processes  described  in  §  138.  The  slide  is  then  drained  and 
put  away  to  dry  until  every  trace  of  water  has  completely 


SERIAL    SECTION    MOUNTING.  341 

evaporated  away  from  under  the  sections.  This  drying 
may  be  performed  at  the  temperature  of  the  laboratory,  in 
which  case  many  hours  will  be  necessary  (to  be  safe  it  will 
generally  be  necessary  to  leave  the  sections  overnight). 
Or  it  may  be  performed  in  a  stove  or  on  a  water-bath,  at  a 
temperature  a  few  degrees  below  the  melting-point  of  the 
paraffin  (best  not  above  40°  C.),  in  which  case  fixation  will  be 
much  more  rapid,  large  thin  sections  being  often  sufficiently 
fixed  in  an  hour,  though  thick  ones  will  require  half  a  dozen 
hours  or  more.  T/ie  paraffin  must  not  be  allowed  to  melt 
before  the  sections  are  perfectly  dry,  the  sections  are  sure  to 
become  detached  if  it  does.  Perfectly  dry  sections  have  a 
certain  brilliant  transparent  look  that  is  easily  recognisable. 
As  soon  as  dry  they  are  perfectly  fixed,  and  the  paraffin 
may  be  removed  and  they  may  be  treated  with  any  desired 
liquids  without  more  risk  of  their  falling  off  than  is  the  case 
with  any  other  mode  of  fixation.  To  remove  the  paraffin 
most  workers  first  melt  it,  and  then  remove  it  with  a  solvent. 
I  find  this  is' by  no  means  necessary,  all  that  is  requisite  is 
to  put  the  slide  into  a  tube  of  xylol,  which  in  a  few  seconds, 
or  minutes  at  most,  removes  the  paraffin  perfectly  without 
any  heat. 

(b)  For  series  of  numerous  small  sections.  Clean  a  slide 
perfectly,  so  that  water  will  spread  on  it  without  any  ten- 
dency to  run  into  drops  (see  p.  142).  Breathe  on  it,  and 
with  a  brush  draw  on  it  a  streak  of  water  as  wide  as  the 
sections  and  a  little  longer  than  the  first  row  of  sections 
that  it  is  intended  to  mount.  With  a  dry  brush  arrange 
the  first  row  of  sections  (which  may  be  either  loose  ones  or 
a  length  of  a  ribbon)  on  this  streak.  Breathe  on  the  slide 
again,  draw  on  it  another  streak  of  water  under  the  first  one 
and  arrange  the  next  row  of  sections  on  it,  and  so  on  until 
the  slide  is  full.  Then  breathe  on  the  slide  again,  and  with 
the  brush  add  a  drop  of  water  at  each  end  of  each  row  of 
sections,  so  as  to  enable  them  to  expand  freely ;  then  warm 
the  slide  so  as  to  flatten  out  the  sections,  taking  care  not  to 
melt  the  paraffin.  Some  persons  do  this  by  holding  it  over 
a  small  flame  for  a  few  seconds.  I  prefer  to  keep  a  slab  of 
thickish  glass  in  the  drawer  of  the  water-bath,  so  as  to  have 
it  warm,  and  lay  the  slide  on  it,  watching  the  flattening  of 
the  sections  through  a  lens  if  necessary.  As  soon  as  they 


142  CHAPTER  X. 

are  perfectly  flat,  draw  off  the  excess  of  water  from  one 
corner  of  the  mount  with  a  dry  brush,  and  put  aside  to  dry 
as  before  (a) . 

In  order  to  success  in  this  method  it  is  absolutely  essential 
that  the  sections  be  perfectly  expanded  and  come  into  close 
contact  with  the  slide  at  all  points.  And  to  ensure  this  it 
is  necessary  that  the  slide  should  be  perfectly  free  from 
grease,  so  that  the  water  may  wet  it  equally  everywhere. 
The  test  for  this  is,  firstly,  to  breathe  on  the  slide;  the 
moisture  from  the  breath  should  condense  .  on  it  evenly  all 
over,  and  disappear  evenly.  Secondly,  streaks  of  water 
drawn  on  it  with  a  brush  should  not  run.  It  is  not  always 
easy  to  obtain  a  slide  that  will  fulfil  these  conditions. 

After  slides  have  been  cleaned  by  one  of  the  processes 
given  in  the  Appendix,  Cleaning  Slides  and  Covers,  they 
should  be  rinsed  with  distilled  water  and  preserved  in  90 
per  cent,  alcohol,  from  which  they  should  be  removed  with 
forceps  when  required  for  use — not  with  the  fingers — then 
simply  drained,  or  wiped  with  a  very  clean  cloth.  If  now  a 
slide  will  not  stand  the  breath  test,  place  a  drop  of  water 
on  it  and  rub  it  in  thoroughly  with  a  damp  cloth  and  try 
again.  If  this  does  not  suffice,  take  a  turn  of  a  corner  of 
the  cloth  round  a  finger  and  rub  it  with  a  piece  of  chalk, 
then  damp  the  cloth  and  rub  the  slide  with  it,  finishing  up 
with  a  clean  part  of  the  cloth  and  clean  water  (DE  GROOT, 
loc.  cit.  supra).  If  after  performing  this  operation  twice 
the  slide  still  refuses  to  take  the  water  properly,  it  should 
be  rejected  as  incorrigible  ;  for  there  are  apparently  some 
sorts  of  glass  that  can  never  be  got  to  wet  thoroughly. 

Tap  water  seems  preferable  to  distilled  water ;  it  seems  to 
spread  better  and  give  a  stronger  adhesion.  NUSBAUM  adds 
a  trace  of  gum  arabic  (one  or  two  drops  of  mucilage  to  a 
glass  of  water)  ;  and  APATHY  (Microtechnik,  p.  126)  adds  1 
per  cent,  of  Mayer's  albumen  (next  §). 

Some  workers  have  used  alcohol  (50  per  cent,  or  70  per 
cent.)  instead  of  water  ;  but  this  I  believe  to  be  now  gener- 
ally abandoned. 

This  is  the  most  elegant  method  of  any.  No  cement 
being  employed,  there  is  nothing  on  the  slide  except  the 
sections  that  can  stain,  or  appear  as  dirt  in  the  mount. 
Tissues  do  not  suffer  in  the  least  from  the  drying,  provided 


SERIAL    SECTION    MOUNTING.  143 

tin-  material  has  been  properly  imbedded.  Sections  stick 
so  fast  by  this  method  that  they  will  stand  staining  on  the 
slide  ;  they  will  stand  watery  or  other  fluids  for  weeks,  so 
long  as  they  are  not  alkaline,  as  these  may  cause  them  to 
become  detached.  When  successfully  performed  it  is  quite 
safe,  provided  that  the  sections  are  of  a  suitable  nature. 
They  must  be  such  as  to  afford  a  sufficient  continuous  sur- 
face, everywhere  in  contact  with  the  slide.  Sections  of 
parenchymatous  organs  stick  well ;  sections  of  thin-walled 
tubular  organs  stick  badly,  often  so  badly  that  the  method 
is  really  not  safe  for  them  at  all.  Sections  of  chitinous 
organs  are  very  unsafe.  The  larger  and  thinner  sections 
are,  the  better  do  they  stick,  and  vice  versa.  Sections  from 
material  that  has  been  fixed  in  chromic  or  osmic  mixtures 
are  said  to  adhere  less  well  than  sections  from  alcohol  or 
sublimate  material ;  1  find,  however,  that  mine  adhere  per- 
fectly well  if  otherwise  suitable.  The  method  has  the  dis- 
advantage of  being  lengthy.  And  Mayer  states  that  if  it  is 
employed  with  stained  material,  the  heat  and  drying  may 
attack  some  kinds  of  stains. 

183.  MAYER'S  Albumen  (Mitth.  Zool  Stat.  Neapel,  iv,  1883; 
Journ.  Roy.  Mic.  Soc.  [N.S.],  iv,  1884,  p.  317;  Intemat. 
Mnuatschr.  f.  Anat.,  3887,  Heft  2;  Journ.  Rot/.  Mic.  Soc., 
1888,  p.  160. — White  of  egg,  50  c.c. ;  glycerin,  50  c.c. ;  sali- 
cylate  of  soda,  1  grm.  Shake  them  well  together,  and  filter 
into  a  clean  bottle.  The  filtering  may  take  days  or  a  week, 
but  the  preparation  does  not  spoil  meanwhile. 

I  find  it  convenient  to  beat  up  the  egg  with  a  little  water 
before  adding  the  glycerin  and  filtering,  the  salicylate 
being  dissolved  in  the  water  in  the  first  instance. 

A  very  thin  layer  of  the  mixture  is  spread  on  a  cold  slide 
with  a  fine  brush  and  well  rubbed  in  with  the  finger.  The 
st- i-t ions  are  laid  on  it  and  pressed  down  lightly  with  a  brush. 
The  slide  is  then  warmed  for  some  minutes  on  a  water-bath, 
and  the  paraffin  removed  with  a  solvent. 

It  is  no't  necessary  to  use  a  water-bath  for  warming  the 
slide.  I  prefer  to  warm  for  an  instant  over  a  flame  until 
the  paraffin  melts ;  it  is  then  instantly  removed  by  means  of 
xylol,  tuluol,  or  the  like. 

It  is  not  'necessary  to  warm  the   slide   at  all ;   the  paraffin 


144  CHAPTER    X. 

can  be  removed  in  the  cold  if  desired  by  putting  the  slide  into 
toluol,  xylol,  or  the  like.  But  the  slide  must,  in  any  case, 
be  very  thoroughly  treated  with  alcohol  after  removal  of  the 
paraffin,  in  order  to  get  rid  of  the  glycerin,  which  will  cause 
cloudiness  if  not  perfectly  removed. 

The  function  of  the  glycerin  is  merely  to  keep  the  layer 
of  albumen  moist. 

Miss  A.  M.  CLAYPOLE  has  written  a  paper  complaining  that  the  method 
is  uncertain,  because  too  much  heat  may  injure  the  tissues,  and  if  too  little 
be  applied  the  albumen  will  not  coagulate.  This  is  a  misapprehension. 
No  heat  whatever  is  required  to  coagulate  the  albumen  ;  the  alcohol  will  do 
that  sufficiently. 

This  method  allows  of  the  staining  of  sections  on  the  slide 
with  perfect  safety,  both  with  alcoholic  and  aqueous  stains, 
provided  they  be  not  alkaline. 

This  method  can  be  combined  with  the  water  process  for 
flattening  out  sections  (last  §),  as  described  by  HKNNEGUY 
(Journ.  de  I'Anat.  et  de  la  Physiol.,  1891,  p.  398).  A  drop 
of  water  is  spread  by  means  of  a  glass  rod  on  a  slide  pre- 
pared with  white -of-egg  mixture,  the  sections  are  arranged 
on  it,  the  whole  is  warmed  (not  to  the  melting-point  of  the 
paraffin)  until  the  sections  flatten  out ;  the  water  is  then 
evaporated  off  at  a  temperature  of  about  40°  C.,  and  as  soon 
as  it  has  sufficiently  disappeared,  which  at  thnt  temperature 
will  be  in  about  ten  minutes,  the  paraffin  is  melted,  and  the 
slide  further  treated  as  above  described. 

See  also  the  description  of  this  method  given  by  OHLMACHE-E  (Journ. 
Amer.  Med.  Ass.,  April,  1893),  who  has  independently  worked  out  the 
same  process. 

The  so-oalled  "  Japanese "  method,  attributed  to  IKEDA  by  REIXKE 
(Zeit.f.  wiss.  Mik.,  xii,  1895,  p.  21)  is  merely  that  of  Henneguy. 

According  to  my  experience  the  albumen  method  is  abso- 
lutely safe.  It  has  the  defect  that  certain  plasma  stains 
(not  chromatin  stains)  colour  the  albumen  very  strongly  and 
cannot  be  removed  from  it. 

It  sometimes  happens  that  the  mixture  after  it  has  stood 
for  some  time  becomes  turbid,  a  change  which  has  been 
attributed  to  the  development  of  a  microbe.  I  know  of  no 
means  of  preventing  the  mixture  from  going  bad  in  this  way, 
though  I  have  found  that  it  keeps  better  when  freely 
exposed  to  the  sun.  It  has  been  stated  (VOSSELER,  Zeit.  f. 


SERIAL    SECTION   MOUNTING.  145 

//•/>•*.  Mik.,  vii,  4,  1891,  p.  457)  that  after  a  time  the  mix- 
ture loses  its  adhesive  properties,  and  should  be  thrown 
away  (GRANDIS  also  [Attl  Accad.  Lincei,  Rend.  (4),  vi,  1890, 
p.  138;  Arch.  Ital.  Biol.,  xiv,  1891,  p.  412]  states  that  the 
albumen  of  the  mixture  decomposes  after  a  time).  That  is 
not  my  experience.  I  find  the  liquid  either  first  becomes 
milky,  then  altogether  turbid,  and  at  last  coagulates,  passing 
into  a  caseous  state,  or  it  may  undergo  a  hyaline  coagulation, 
drying  up  like  amber.  But  up  to  the  very  last  it  does  not 
in  the  least  degree  lose  its  adhesive  properties.  As  long  as 
there  is  enough  moisture  in  it  to  moisten  the  brush,  I  have 
always  found  it  to  stick  as  well  as  the  first  day. 

184.  MANN'S  Albumen   Method  (Zeit.  f.   idss.   Mik.,  xi,   4,   1894, 
p.  486). — Shake  up  white  of  egg  with  ten  volumes  of  distilled  water  and 
filter  twice  through  the  same  paper.     Spread  this  on  a  stock  of  slides  with 
a  glass  rod  ;  let  them  drain  and  dry.     Arrange  and  expand  the  sections 
thereon  by  the  water  method,  §  182,  put  the  slide  for  five  minutes  on  a 
stove  heated  to  35°  C.,  then  treat  with  xylol  and  alcohol. 

185.  SCHALLIBAUM'S  Collodion  Method  (Arch.  f.  ntik.  Anat.,  xxii, 
1883,  p.   565). — One  part  of   collodion  is  shaken  up  with  three  to  four 
volumes  (according  to  the  consistency  of  the  collodion)  of  clove  oil  or 
lavender  oil.     This  should  give  a  clear  solution.     A  little  is  spread  thinly 
on  a  slide  with  a  small  brush.     After  arranging  the  sections  on  the  pre- 
pared surface,  warm  over  a    water-bath,  gently,  until  the  clove   oil   has 
evaporated  (five  to  ten  minutes).     The  sections  are  then  found  to  be  fixed, 
and,  it  is  said,  can  be  treated  for  days  with  turpentine,  chloroform,  alcohol, 
and  watery  fluids,  without  becoming  detached,  thus  allowing  staining  on 
the  slide.     According  to  my  experience,  however,  the  method  is  certainly 
not  safe  for  that  purpose,  and  should  be  discarded  in  favour  of  the  water  or 
the  albumen  method,  and  should  only  be  used  for  already  stained  sections. 
See,  however,  further  details  in  previous  editions,  or  RABL  (Zeit.f.  wiss. 
Mile.,  xi,  2,  p.  179) ;  FIELD  and  MABTIN  (Bull.  Soc.  Zool.  de  France,  xix, 
1894,  p.  48)  j  GALLEMAEBTS  (Bull.  Soc.  Beige  de  Micro.,  xv,  1889,  p.  56 ; 
Zeit.  f.  wiss.  Mik.,  vi,  p.  4,  1889,  p.  493);  SUMMEBS  (Amer.  Mon.  Mic. 
Journ.,  1887,  p.  73  ;  Zeit.  f.  wiss.  Mik.,  iv,  4,  1887,  p.  482) ;   STBASSEB 
(Zeit.f.  unss.  Mik.,  iv,  1,  1887,  p.  45). 

186.  STBASSEB'S  Collodion-Paper  Method  (Zeit.f.  iciss.  Mik.,  iii,  3, 
1886,  p.  316). — This  is  an  extremely  complicated  modification  of  Weigert's 
method  for  celloidin  sections,  and  is  only  adapted  for  use  with  STBASSEB'S 
automatic  ribbon-microtome.     See  the  original  papers  in  Zeit.f.  wiss.  Mik., 
iii,  3,  1886,  p.  316 ;   vi,  2,  1889,  p.  154  ;  vii,  3,  1890,  p.  290 ;  ibid.,  p.  304  ; 
ix,  1,  1892,  p.  8 ;  Journ.  Roy.  Mic.  Soc.,  1892,  p.  703  ;  Zeit.f.  wiss.  Mik., 

10 


146  CHAPTER    X. 

xii,  2,  1895,  p.  154  (Journ.  Roy.  Mic.  Soc..  1895,  p.  702) ;  Zeit.f.  wiss.  Mik., 
xiv,  1,  1897,  p.  39. 

187.  The  Shellac  Method  (GIESBRECHT,  Zool  Am.,  1881,  p.  484).— 
Prepare  a  stock  of  slides  covered  with  a  thin  and  even  film  of  shellac.     This 
is  done  as  follows  : — Make  a  not  too  strong  solution  of  brown  shellac  in  abso- 
lute alcohol,  filter  it  thoroughly  ;  warm  the  slides,  and  spread  over  them  a 
layer  of  shellac  by  means  of  a  glass  rod  dipped  in  the  solution  and  drawn 
once  over  each  slide.     Let  the  slides  dry. 

You  may  now  either  take  a  prepared  slide  and  brush  it  over  very  thinly 
with  creasote  applied  by  means  of  a  brush,  and  arrange  the  sections  on  the 
sticky  surface ;  then  heat  the  slide  on  a  water-bath  for  about  a  quarter  of 
an  hour  at  the  melting-point  of  the  paraffin — the  slide  is  allowed  to  cool, 
and  the  paraffin  is  dissolved  away  bj  dropping  turpentine  on  to  the  sections, 
which  are  then  mounted  in  Canada  balsam.  Or  (MAYER,  Intern.  Monatsschr. 
/.  Anal.,  etc.,  1887,  Heft  2 ;  Zeit.  f.  wiss.  Mile.,  iv,  1,  1887,  p.  77)  the 
brushing  with  creasote  is  omitted  and  the  sections  are  arranged  on  the  dry 
film  and  gently  pressed  down  on  to  it,  then  exposed  for  half  a  minute  to 
vapour  of  ether,  which  fixes  them. 

The  method  does  not  allow  of  staining  on  the  slide,  and  is  in  my  opinion 
superfluous,  but  see  further  details  in  previous  editions,  or  Mitth.  d.  Zool. 
Stat.,\u,  1881,  p.  148  ;  Journ.  Roy.  Mic.  Soc.  (N.  S.),  vol.  ii,  1882,  p.  888  ; 
WHITMAN'S  Methods  in  Microscopical  Anat.,  p.  117 ;  CALDWELL,  Quart. 
Journ.  Mic.  Soc.  (N.  S.),  Ixxxvii,  1882,  p.  336  ;  P.  MAYER,  Amer.  Natural, 
1882,  p.  733 ;  Zeit.f.  wiss.  Mik.,  iv,  1,  1887,  p.  77 ;  Journ.  Roy.  Mic.  Soc., 
1885,  p.  910 ;  and  LEE  und  MAYER,  Gh'undzilge,  etc.,  1898,  p.  118. 

188.  VAN  WALSEM'S  Gelatin  Process.— Extremely  complicated.     See 
Zeit.  f.  wiss.  Mik.,  xi,  2,  1894,  pp.  229  to  235,  or  Journ.  Roy.  Mic.  Soc., 
1895,  p.  122. 

189.  OBREGIA'S  process  given  below,  §  196,  for  celloidm 
sections,  is  also  applicable  to  paraffin  sections. 

For  BLOCHMAN'S  modification  of  Weigert's  process,  by  means  of  which 
large  sections  can  be  preserved  unmounted,  see  Zeit.  f.  iviss.  Mik.,  xiv,  2, 
1897,  p.  189. 

For  other  methods  with  gum,  gelatin,  etc.,  suppressed  as  superfluous,  see 
previous  editions. 


Methods  for  Watery  Sections. 

190.  Foi/s  Gelatin  (FoL,  Luhrl.,  p.  132). — Four  grammes 
of  gelatin  are  dissolved  in  20  c.c.  of  glacial  acetic  acid  by 
heating  on  a  water-bath  and  agitation.  To  5  c.c.  of  the 
solution  add  70  c.c.  of  70  per  cent,  alcohol  and  1  to  2  c.c,  of 
5  per  cent,  aqueous  solution  of  chrome-alum.  Pour  the  mix- 


SERIAL    SECTION    MOUNTING.  147 

ture  on  to  the  slide  and  allow  it  to  dry.  In  a  few  hours  the 
gelatin  passes  into  the  insoluble  state.  It  retains,  however, 
the  property  of  swelling  and  becoming  somewhat  sticky  in 
presence  of  water.  The  slide  may  then  be  immersed  in 
\\ator  containing  the  sections  ;  these  can  be  slid  into  their 
places,  and  the  whole  lifted  out  :  the  sections  will  be  found 
to  be  fixed. 

This  method  is  specially  intended  for  sections  made  under 
water,  large  celloidin  sections  amongst  others. 


for  Celloidin  Sections. 

191.  The  Albumen  Method.—  I  find    that   celloidin    sections 
may  be  mounted  on  Mayer's  albumen,  and  have  the  celloidin 
removed,  if  desired,  by  putting  them  into  ether-alcohol.    Care 
must  be  taken  to  press  them  down  very  thoroughly  on  to  the 
albumen. 

So  also  JORDAN  (Zeit.  f.  wins.  Mik.,  xv,  1,  1898,  p.  54; 
Joum.  Roij.  Mic.  Soc.,  1898,  p.  600),  who  coagulates  the 
albumen  by  heat,  the  sections  being  covered  with  a  layer  of 
tissue-paper  and  a  second  slide  over  it,  to  prevent  them  from 
drying  through  the  heat. 

192.  SOMMERS'  Ether  Method  (Amer.  Hon.  Mic.  Journ.,  1887, 
p.  7:5;  Zeit.  f.  wis*.  Mile.,  iv,  4,    1887,  p.  482;  Journ.  Roy. 
Mi<\  Soc.,  1887,  p.  523).  —  Place  the  sections  in  95  per  cent. 
alcohol  for  a  minute  or  two,  arrange  on  the  slide,  and  then 
pour  over  the  sections  sulphuric  ether  vapour,  from  a  bottle 
partly  full  of  liquid  ether.      The  celloidin  will  immediately 
soften  and  become  perfectly  transparent.      Place  the  slide  in 
80  per   cent,   alcohol,  or   even   directly  in   95    per  cent,  if 
desired.      The  sections,  it  is  said,  will  be  found  to  be  firmly 
fixed,  and  may  be  stained  if  desired.      I  have  not  myself 
found  this  method  safe. 

SCHIEFFERDECKER  (Zeit.  f.  wi**.  Mil'.,  v,  4,  1888,  p.  507) 
recommends  that  the  slide  be  one  that  has  been  previously 
prepared  with  a  layer  of  collodion  if  it  is  desired  to  stain  on 
the  slide  ;  but  if  not  a  clean  slide  is  perfectly  sufficient.  The 
^lide  may,  of  course,  be  treated  with  ether  vapour  in  a  prepa- 
ration glass  or  similar  arrangement. 

GAGE  (Proc.  Amer.  Soc.  Mic.,  1892,  p.  82)  advises  that  the 


148  CHAPTER  X. 

slide  be  one  that  has  been  previously  coated  with  a  0*5  per 
cent,  solution  of  white  of  egg  and  dried ;  the  collodion 
adheres  much  more  strongly  to  an  albuminised  surface. 

AUBUETIN  (Anat.  Anz,,  xiii,  1897,  p.  90;  Journ.  Roy.  Mic.  Soc.,  1897, 
p.  174)  arranges  on  a  clean  slide,  dehydrates  the  sections  with  blotting  paper 
and  treatment  with  absolute  alcohol,  then  drops  on  to  them  a  mixture  of 
alcohol  and  ether  which  dissolves  out  the  celloidin  from  the  sections,  then 
allows  the  thin  collodion  thus  formed  to  evaporate  into  a  thin  sheet  on  the 
slide.  Then  70  per  cent,  alcohol  and  other  desired  reagents. 

193.  APATHY'S  Oil  of  Bergamot  Method  (Mitth.  Zool.  Stat. 
Neapel,  1887,  p.  742 ;  Zeit.  f.  wiss.  Mik.,  v,  1,  1888,  p.  46, 
and  v,  3,  1888,  p.  360;  Journ.  Roy.  Mic.  Soc.,  1888,  p.  670). 
— Cut  with  a  knife  smeared  with  yellow  vaselin  and  wetted 
with  95  per  cent,  alcohol.  Float  the  sections,  as  cut,  on 
bergamot  oil  (must  be  green,  must  mix  perfectly  with  90  per 
cent,  alcohol,  and  must  not  smell  of  turpentine),  or  011  carbol- 
xylol  (Mikroteclmik,  p.  176).  The  sections  spread  them- 
selves out  on  the  surface  of  the  oil ;  before  they  sink  each 
one  is  pushed  by  means  of  a  needle  into  its  place  on  a  slip 
of  tracing-paper  dipped  into  the  oil.  When  the  requisite 
number  of  sections  have  been  arranged  on  the  paper,  you 
drain  the  paper,  dry  the  under  side  of  it  with  blotting-paper, 
turn  it  over,  and  gently  press  it  down  with  blotting-paper 
on  to  a  carefully  dried  slide.  Kemove  the  paper  by  rolling- 
it  up  from  one  end.  The  sections  remain  adhering  to  the 
slide,  and  may  have  the  remaining  bergamot  oil  removed 
from  them  by  means  of  a  cigarette  paper.  If  they  are 
already  stained,  nothing  remains  but  to  add  balsam  and  a 
cover. 

In  the  case  of  unstained  or  very  small  objects  it  is  well  to  add  a  little 
alcoholic  solution  of  safranin  to  the  bergamot  oil.  The  celloidin  of  the  sec- 
tions  becomes  coloured  in  it  in  a  few  seconds,  and  makes  them  readily  visible. 
The  colour  disappears  in  a  few  days  after  mounting. 

The  process  may  be  much  simplified  (APATHY,  Mikro- 
technik,  p.  127)  by  omitting  the  arrangement  on  the  paper 
and  transferring  the  sections  direct  from  the  bergamot  oil  to 
the  slide,  which  (ibid.,  p.  176)  may  have  been  previously 
collodionised  and  dried.  The  function  of  the  bergamot  oil 
is  to  flatten  out  the  sections. 

If  the  sections  are  to  be  stained,  the  slide  after  removal 


SERIAL   SECTION   MOUNTING.  149 

of  the  bergamot  oil  is  exposed  for  a  few  minutes  to  the 
vapour  of  a  mixture  of  ether  and  alcohol,  then  brought  into 
90  per  cent,  alcohol,  and  after  a  quarter  of  an  hour  therein 
may  be  stained  in  any  fluid  that  contains  70  per  cent,  alcohol 
or  more. 

If  it  be  desired  to  stain  in  a  watery  fluid,  care  must  have 
been  taken  when  arranging  the  sections  to  let  the  celloidin 
of  each  section  overlap  that  of  its  neighbours  at  the  edges, 
so  that  the  ether  vapour  may  fuse  them  all- into  one  con- 
tinuous plate.  This  will  become  detached  from  the  slide  in 
watery  fluids,  and  may  then  be  treated  as  a  single  section. 

194.  APATHY'S  Series-on-the-Knife  Method  (Zeit.f.wiss.Mik., 
vi,  2,  1888,  p.  168). — The  knife  is  well  smeared  with  yellow 
vaselin  rubbed  evenly  on  with  the  finger,  and  is  wetted  with 
alcohol  of  70  to   90  per  cent.      As  fast  as  the  sections  are 
cut  they  are  drawn  with  a  needle  or  small  brush  to  a  dry 
part  of  the  blade,  and  there  arranged  in  rows,  the  celloidin 
of  each  section  overlapping  or  at  least  touching  that  of  its 
neighbours.      The  rows  are  the  length  of  the  cover-glass,  and 
are  arranged  one  under  the   other  so  as  to  form  a  square  of 
the  size  of  the  cover-glass.      When  a  series  (or  several  series, 
if  you  like)  has  been  thus  completed,  the  sections  are  dried 
by  laying  blotting-paper  on  them   (there  is  no  risk  of  their 
becoming   attached   to   it,   as  they   are    held   down  by    the 
vaselin).      The  series  is  then  painted  over  with  some  of  the 
thickest  celloidin  solution  used  for  imbedding,  is  allowed  to 
evaporate   for  five   minutes  in   the  air,   and  is  then   either 
wetted   with    70   per   cent,   alcohol,   and  allowed  to  remain 
whilst  cutting  is  proceeded  with,  or  (if  no  more  sections  are 
to  be  cut,  or  if  the  knife  is  now  full)    the  knife  is  removed 
and    brought   for   half  an   hour   into    70   per  cent,   alcohol. 
This  hardens  the  celloidin  around  the  sections  into  a  con- 
tinuous lamella,  which  can  be  easily  detached  by  means  of  a 
<<-al])el,  and  stained,  or  further  treated  as  desired.    It  is  well 
to  bring  it  at  once   on  to  a  slide,  moisten  the  edges  of  the 
Celloidin  plate  with  ether  and  alcohol  mixture,  so  that  it  may 
not  become  detached,  and  bring  the  whole  into  the  staining 
solution. 

195.  WEIGERT'S  Collodion  Method  (Zeit.  f.   «rw*.  J/Vfc.,  1885, 
p.  490). — Sections  are  cut  wet  with  alcohol.      Care  should 


150  CHAPTER   X. 

be  taken  not  to  have  so  much  alcohol  on  the  knife  as  to  cause 
the  sections  to  float.  Prepare  a  slip  of  porous  but  tough 
paper  (Weigert  recommends  "  closet  paper ")  of  about 
twice  the  width  of  the  sections.  Soak  it  in  alcohol,  take  it 
by  both  ends,  stretch  it  slightly,  and  lower  it  on  to  the 
section  that  is  on  the  knife.  The  section  will  adhere  to  the 
paper,  and  is  taken  up  by  moving  the  slip  horizontally  or 
slightly  upwards,  away  from  the  edge  of  the  knife.  Take 
up  the  first  section  towards  the  end  of  the  paper  that  you 
hold  in  your  left  hand,  and  let  the  remaining  sections  follow 
in  order  from  left  to  right.  After  each  section  has  been 
taken  up,  the  slip  is  placed,  whilst  the  next  section  is  being 
cut,  with  the  sections  upwards  on  a  moist  surface  prepared 
by  arranging  several  layers  of  blotting-paper,  covered  with 
one  layer  of  closet  paper,  in  a  plate,  and  saturating  the 
whole  with  alcohol.  When  all  the  sections  have  been 
arranged  on  the  slip,  you  pass  to  the  next  stage  of  the  pro- 
cess, the  collodionisation  of  the  series. 

This  is  done  in  two  steps.  The  first  of  these  consists  in 
transporting  the  series  on  to  a  plate  of  glass  prepared  with 
collodion.  The  plate  is  prepared  beforehand  by  pouring  011 
to  it  collodion  and  causing  it  to  spread  out  into  a  thin  layer, 
as  photographers  do,  and  allowing  it  to  dry.  (A  number  of 
the  plates  may  be  prepared  and  kept  indefinitely  in  stock ; 
microscope  slides  will  do  for  series  of  small  sections.)  Take 
one  of  these  plates  ;  lay  the  slip  of  paper  with  the  sections 
on  the  plate,  the  sections  downwards  ;  press  it  down  gently 
and  evenly,  and  the  sections  will  adhere  to  the  collodion ; 
then  carefully  remove  the  paper.  (Do  not  place  more  than 
one  or  at  most  two  lines  of  sections  on  the  same  plate,  for 
those  first  placed  run  the  risk  of  becoming  dry  whilst  you 
are  placing  the  others.)  This  finishes  the  first  stage  of  the 
collodionising  process. 

Now  remove  with  blotting-paper  any  excess  of  alcohol  that 
may  remain  on  or  around  the  sections,  pour  collodion  over 
them,  and  get  it  to  spread  in  an  even  layer.  As  soon  as  this 
layer  is  dry  at  the  surface  you  may  write  any  necessary  indi- 
cations on  it  with  a  small  brush  charged  with  methylen  blue 
(the  colour  will  remain  fast  throughout  all  subsequent 
manipulations) . 

The  plate  may  now  either  be  put  away  till  wanted  in  80 


SERIAL    SECTION  MOUNTING.  151 

per  cent,  alcohol,  or  may  be  brought  into  a  staining  fluid. 
The  watery  fluid  causes  the  double  sheet  of  collodion  to 
become  detached  from  the  glass,  holding  the  sections  fast 
between  its  folds.  It  is  then  easy  to  stain,  wash,  dehydrate, 
and  mount  in  the  usual  way,  merely  taking  care  not  to  use 
alcohol  of  more  th«n  90  to  96  per  cent,  for  dehydration. 
Weigert  recommends  for  clearing  the  mixture  of  xylol  and 
carbolic  acid  (§  161). 

The  series  should  be  cut  into  the  desired  lengths  for 
mounting  whilst  in  the  alcohol.  It  is  perhaps  safer  to  lay 
them  out  for  cutting  on  a  strip  of  closet  paper  saturated  with 
alcohol. 

A  good  method  for  large  and  thick  sections  that  do  not 
require  flattening,  not  for  series  of  small  thin  ones. 

It  is  suggested  by  STRASSER  that  gummed  paper  might  be  an  improve- 
ment on  the  glass  plates  used  in  this  process — especially  for  very  large 
sections. 

The  modification  of  .Weigert 's  method  proposed  by  WINTEBSTEINEB  (Zeit. 
f.  iviss.  Mik.,  x,  3,  1893,  p.  316)  consists  in  suppressing  the  alignment  of 
the  sections  on  the  strip  of  paper,  and  slipping  them  direct  from  the  knife 
on  to  the  prepared  glass. 

196.  OBEEGIA'S  Method  for  Paraffin  or  Celloidin  Sections. — 
This  method  was  originally  described  in  the  Neurologisclies 
C>  utralb.,  ix,  1890,  p.  295,  and  is  given  in  the  third  edition 
of  WOODHKAD'S  Practical  Pathology.  It  is  described  with 
modifications  by  GULLAND,  Joum.  of  Path.,  February,  1893. 
Slides,  or  glass  plates  of  any  size,  are  coated  with  a  solution 
made  of — 

Syrupy   solution   of    powdered   candy- 
sugar    made   with    boiling   distilled 
water            .          .          .          .          .30  c.c. 
95  per  cent,  alcohol.           .           .  20    „ 
Transparent    syrupy   solution   of   pure 
dextrin  made   by  boiling  with   dis- 
tilled water 10    „ 

They  are  dried  slowly  for  two  or  three  days  until  the 
surface  is  just  sticky  to  the  moist  finger.  Paraffin  sections 
are  arranged  and  heated  for  a  few  minutes  to  a  temperature 
slightly  above  the  melting-point  of  the  paraffin.  The  paraffin 
is  removed  by  some  solvent,  such  as  xylol  or  naphtha,  and 


152  CHAPTER    X. 

this  is  in  turn  removed  by  absolute  alcohol.  The  alcohol  is 
poured  off,  and  the  sections  are  covered  with  solution  of 
celloidin  or  with  a  solution  of  3  per  cent,  of  photoxylin  or 
Sobering' s  "  Celloidinwolle  "  in  a  mixture  of  equal  parts  of 
ether  and  absolute  alcohol.  The  plates  are  left  to  evaporate 
for  ten  minutes  in  a  horizontal  position,  then  brought  into 
water,  in  which  the  sheet  of  celloidin  with  the  sections  soon 
becomes  detached,  and  may  be  further  treated  as  desired, 
e.  g.  as  in  Weigert's  process,  §  195.  (It  is  well  to  divide  the 
sheet  of  celloidin  into  ribbons  by  running  the  point  of  a 
knife  down  it  as  soon  as  evaporation  has  produced  a  very 
slight  solidification,  and  the  evaporation  must  not  be  artifi- 
cially hastened.) 

This  is  the  process  for  paraffin  sections  ;  for  celloidin  sec- 
tions the  sections  are  taken  up  in  order  on  a  strip  of  paper 
(glossed  tissue  paper,  satinisirtes  Seid&npapierj  the  sections  to 
be  011  the  glossed  side)  as  in  WEIGEKT'S  method,  and  laid 
down  on  the  glass  in  the  same  way,  and  then  covered  with 
the  celloidin  or  photoxylin  solution  and  evaporated  as  de- 
scribed. The  advantage  of  Obregia's  process  is  that  it  is 
equally  applicable  to  paraffin  sections,  to  celloidin  sections, 
and  to  sections  of  material  that  has  not  been  imbedded  at  all. 

DIMMER  (Zeit.f.  wiss.  Mik.,  xvi,  1,  1899,  p.  44;  Journ.  Roy.  Mic.  Soc., 
1899,  p.  448)  coats  the  slides  with  a  solution  of  about  16  parts  of  gelatin 
in  300  of  warm  water,  and  dries  them  (two  days),  and  proceeds  in  other 
respects  as  above. 

197.  GIACOMINI'S  collodion-gelatin  process  for  large  sections,  see  Gazzetta 
delle  Cliniche,  November,  1885,  Zeit.  f.  wiss.  Mile.,  1885,  p.  531,  or  the 
first  edition  of  the  Traite  of  LEE  et  HENNEGUY,  p.  392. 


CHAPTER   XI. 
STAINING. 

198.  The  Kinds  of  Stains. — Stains  are  either  General  or 
Special  (otherwise  called  Specific,  or  Selective,  or  Elective). 
A  general  stain  is  one  that  takes  effect  on  all  the  elements  of 
a  preparation.  A  special,  specific,  selective,  or  elective  stain 
is  one  that  takes  effect  only  on  some  of  them,  certain  elements 
being  made  prominent  by  being  coloured,  the  rest  either 
remaining  colourless  or  being  coloured  with  a  different 
intensity  or  in  a  different  tone.  To  obtain  this  differentiation 
i<  the  chief  object  for  which  colouring  reagents  are  employed 
in  microscopic  anatomy. 

Two  chief  kinds  of  this  selection  may  be  distinguished, — 
kittologiuil  selection  and  cytological  selection.  In  the 
former  an  entire  tissue  or  group  of  tissue  elements  is  promi- 
nently stained,  the  elements  of  other  sorts  present  in  the 
preparation  remaining  colourless  or  being  at  all  events 
differently  stained,  as  in  a  successful  impregnation  of  nerve- 
endings  by  means  of  gold  chloride.  This  is  the  kind  of 
stain  that  is  generally  meant  by  a  specific  stain.  In  the 
latter  the  stain  seizes  on  one  of  the  constituent  elements  of 
cells  in  general,  for  instance  either  on  the  chromatin  of  the 
nucleus,  or  on  one  or  other  of  the  elements  that  go  to  make 
up  the  cytoplasm. 

Stains  that  thus  exhibit  a  selective  affinity  for  the  sub- 
stance of  nuclei — nuclear  or  chromatin  stains — form  at  present 
the  most  important  class  of  stains  for  the  embryologist  or 
zootomist.  What  the  zootomist  or  embryologist  wants,  in  the 
great  majority  of  cases,  is  not  so  much  to  differentiate  the 
intimate  structures  of  cells  by  means  of  a  colour  reaction,  in 
order  to  study  them  for  their  own  sakes,  as  the  cytologist 
does,  as  merely  to  have  the  nuclei  of  tissues  marked  out  by 


154  CHAPTER    XI. 

staining  in  the  midst  of  the  unstained  material  in  such  a 
way  that  they  may  form  landmarks  to  catch  the  eye,  which 
is  then  able  to  follow  out  with  ease  the  contours  and  rela- 
tions of  the  elements  to  which  the  nuclei  belong ;  the  extra  - 
nuclear  parts  of  these  elements  being  expressly  left  unstained 
in  order  that  as  little  light  as  possible  may  be  absorbed  in 
passing  through  the  preparation.  Possibly  this  may  be  an 
irrational  procedure,  but  it  is  found  in  practice  to  be  very 
efficient  for  general  work. 

To  these  must  be  added  another  group  of  stains  of  the 
greatest  importance  to  the  cytologist  and  histologist,  the 
pla*matic  stains,  or  plasma  stains.  These  take  effect 
especially  on  elements  of  cells  and  tissues  other  than  the 
chromatin — for  instance,  011  the  reticulum  of  cytoplasm,  or 
on  its  granules,  or  on  polar  corpuscles,  etc.,  or  on  the  formed 
material  of  tissues — the  chromatin  being  left  as  far  as  possible 
unstained,  in  order  that  it  may  be  counterstamed  in  another 
colour  by  means  of  one  of  the  above-mentioned  chromatin 
stains. 

In  this  book,  therefore,  stains  are  looked  upon  as  being 
(1)  General  stains  ;  (2)  Selective  stains  ;  the  latter  group 
being  subdivided  into  (a)  Nuclear,  (I)  Plasmatic,  (c)  Histo- 
logically  Selective,  or  Specific. 

199.  The  Methods  of  Staining.— Colouring  matters  possessing 
so  great  an  affinity  for  certain  elements  of  tissues  that  they 
may  be  left  to  produce  the  desired  electivity  of  stum  without 
any  special  manipulation  on  the  part  of  the  operator,  are  un- 
fortunately rare.  In  practice,  selective  staining  is  arrived  at 
in  two  ways.  In  the  one,  which  may  be  called  the  progressive 
or  direct  method,  you  make  use  of  a  colouring  reagent  that 
stains  the  elements  desired  to  be  selected  more  quickly  than 
the  elements  you  wish  to  have  unstained  ;  and  you  stop  the 
process  and  fix  the  colour  at  the  moment  when  the  former 
are  just  sufficiently  stained,  and  the  latter  not  affected  to  an 
injurious  extent,  or  not  affected  at  all,  by  the  colour.  This 
is  what  happens,  for  instance,  when  you  stain  the  nuclei  of  a 
preparation  by  treatment  with  very  dilute  alum  haematoxylin  : 
you  get,  at  a  certain  moment,  a  fairly  pure  nuclear  stain  ; 
but  if  you  were  to  prolong  the  treatment,  the  extra-nuclear 
elements  would  take  up  the  colour,  and  the  selectivity  of  the 


STAINING.  155 

stain  would  be  lost.  It  may  be  noted  of  this  method  that  it 
is  in  general  the  one  employed  for  the  colouring  of  specimens 
•/;/  luill:, — a  procedure  which  is  not  possible  with  most  of  the 
reirressive  stains.  It  is  the  old  method  of  carmine  and 
hsematoxylin  staining. 

The  second,  the  regressive  or  indirect  method,  is  the  method 
of  overstating  followed  by  partial  discoloration.  You  begin 
l>y  staining  all  the  elements  of  your  preparation  indis- 
criminately, and  you  then  wash  out  the  colour  from  all  the 
elements  except  those  which  you  desire  to  have  stained, 
these  retaining  the  colour  more  obstinately  than  the  others 
in  virtue  of  a  certain  not  yet  satisfactorily  explained  affinity. 
This  is  what  happens,  for  instance,  when  you  stain  a  section 
of  one  deep  red  in  all  its  elements  with  safranin,  and  then, 
treating  it  for  a  few  seconds  with  alcohol,  extract  the  colour 
from  all  but  the  chromatin  and  nucleoli  of  the  nuclei.  It  is 
in  this  method  that  the  coal-tar  colours  find  their  chief 
employment.  It  is  in  general  applicable  only  to  sections,  and 
not  to  staining  objects  in  bulk  (the  case  of  borax  carmine  is 
an  exception).  It  is  a  method,  however,  of  very  wide 
applicability,  and  gives,  perhaps,  the  most  brilliant  results 
that  have  hitherto  been  attained. 

200.  The  State  of  the  Tissues  to  be  stained. — It  is  generally 
found  that  precise  stains  can  only  be  obtained  with  carefully 
fixed  (i.e.  hardened)  tissues.  Dead,  but  not  artificially 
hardened  tissues  stain  indeed,  but  not  generally  in  a  precise 
manner.  Living  tissue  elements  in  general  do  not  stain  at 
all,  but  resist  the  action  of  colouring  reagents  till  they  are 
killed  by  them  (see,  however,  next  section). 

It  appears  probable,  as  was  first  pointed  out,  I  believe,  by 
PALL  MAYEK,  that  the  usual  histological  stains  obtained  with 
fixed  tissues  are  brought  about  in  two  ways.  Either  they 
result  from  the  combination  of  the  colouring  agent  with 
certain  organic  or  inorganic  salts, — phosphates,  for  instance, 
that  existed  in  the  tissue  elements  during  life  and  were  thrown 
down  in  situ  by  the  fixing  or  hardening  agent  employed,  as 
M-eins  to  happen  when  such  a  fixing  agent  as  alcohol  is  em- 
ployed. Or  they  result  from  the  combination  of  the  colour- 
ing agqiit  with  certain  compounds  that  did  not  pre-exist  in 
tho  tissues,  but  were  formed  by  the  combination  of  the  con- 


156  CHAPTER   XI. 

stitueiits  of  the  tissues  with  the  chemical  elements  brought 
to  them  by  the  fixing  agent,  as  seems  to  happen  when  such 
a  fixing  agent  as  chromic  acid  is  employed — the  compounds 
in  question  being  probably  chiefly  metal  albuminates. 
These  considerations  will  serve  to  show  to  how  great  an 
extent  the  quality  of  a  stain  is  dependent  on  the  nature  of 
the  previous  treatment  the  tissues  have  undergone. 


200  a.  The  Molecular  Processes  involved  in  Staining. — The 
question  whether  the  phenomena  of  staining  and  of  industrial  dyeing  are 
of  a  chemical  order,  as  held  by  some,  or  of  a  purely  physical  order,  as  held 
by  others,  is  outside  the  province  of  this  book.  See  the  elaborate  discussion 
of  the  subject  in  FISCHER'S  Fixirung,  Fdrbung  und  Ban  des  Protoplasmus, 
Jena,  G.  Fischer,  1889. 


201.  Staining  "  intravitam." — Some  few  substances  possess 
the  property  of  staining — or  rather,  tiiigeing — living  cells 
without  greatly  impairing  their  vitality.  Such  are — in  very 
dilute  solutions — cyanin  (or  quinolem),  methylen  blue, 
Bismarck  brown,  anilin  black,  Congo  red,  neutral  red,  Nile 
blue,  and,  under  certain  conditions,  dahlia  and  eosin,  gentian 
violet,  with  perhaps  methyl  violet,  and  some  others  whose 
action  is  not  yet  sufficiently  established  by  experiment. 

As  to  the  employment  of  these  reagents,  it  may  be  noted 
that  they  must  be  taken  in  a  state  of  considerable  dilution, 
and  in  neutral  or  feebly  alkaline  solution — acids  being  of 
course  toxic  to  cells.  Thus  employed,  they  will  be  found  to 
tinge  with  colour  the  cytoplasm  of  certain  cells  during  life ; 
never,  so  far  as  I  can  see,  nuclear  chromatin  during  life  ; — 
if  this  stain,  it  is  a  sign  that  death  has  set  in.  The  stain  is 
sometimes  diffused  throughout  the  general  substance  of  the 
cytoplasm,  sometimes  limited  to  certain  granules  in  it,  which 
have  been  taken,  in  some  cases  certainly  without  sufficient 
reason,  to  be  identical  with  the  granules  of  Altmann 
(Altmann's  Studien  uber  die  Zelle,  1886). 

It  has  been  asserted  by  some  observers  that  the  nucleus 
may  be  stained  during  the  life  of  the  cell  by  means  of 
Bismarck  brown,v  Congo  red,  methylen  blue,  neutral  red,  Nile 
blue,v  and  safranin/  But  it  is  by  no  means  clear  from  the 
statements  of  these  writers  that  the  coloration  observed  by 
them  is  localised  in  the  chromatin  of  the  nucleus.  It  would 


STAINING.  157 

rather  appear  to  be  a  diffuse  coloration  of  the  nuclear  sub- 
stance, which  is  a  very  different   thing. 

I  have  myself  made  a  considerable  number  of  observations 
on  the  subject  of  intra-vitam  staining,  and  have  come  to  the 
same  conclusion  as  GALEOTTI  (Zeif.  f.  u-iss.  Mik.,  xi,  2,  1894, 
p.  172),  namely  that  the  so-called  "  intra-vitam"  stains  are 
imt  true  stains  at  all.  The  diffuse  coloration  above  men- 
tinned  appears  always,  if  the  cell  that  shows  it  has  remained 
in  a  state  of  unimpaired  vitality,  to  be  due  to  simple  absorp- 
tion or  imbibition  of  the  colouring  matter  by  the  cell,  not  to 
a  molecular  combination  of  the  colouring  matter  with  any  of 
the  constituents  of  the  cells.  If  a  cell  thus  coloured  be 
transported  into  a  medium  free  from  the  colouring  matter  it 
will  give  up  unchanged  the  colour  it  had  imbibed,  which 
seems  to  be  a  sufficient  proof  that  the  colouring  matter  had 
not  entered  into  any  molecular  combination  with  the  elements 
of  the  cell,  but  was  simply  loosely  held  in  a  mechanical  way 
in  the  interstices  of  its  substance.  If,  on  the  other  hand,  there 
lias  been  produced  the  above-mentioned  coloration  of  certain 
granules  or  other  cell-contents,  it  is  possible  that  this  may 
be  a  true  stain  in  the  sense  of  being  such  a  combination  as 
is  formed  infixed  material  when  stained.  It  may  be  so,  but 
it  certainly  is  not  always  so,  as  may  sometimes  be  proved 
with  the  greatest  ease  by  putting  the  cell  into  a  colourless 
medium  and  observing  the  supposed  stain  disappear.  And 
in  cases  in  which  this  does  not  happen,  in  which,  there- 
fore, a  more  or  less  fast  stain  has  been  obtained,  it  is 
invariably  found  that  the  stain  is  limited  to  cell-contents  that 
do  not  form  an  integral  part  of  the  living  texture  of  the 
cell ;  the  cell  itself  may  be  living,  but  they  are  not.  These 
granules  or  other  cell-contents  may  be  granules  formed  of 
substances  that  have  been  absorbed  by  the  cell  from  with- 
out— f ood- granules ;  or  they  may  be  katabolic  products,  con- 
n-ting of  matter  that  is  no  longer  alive  and  is  destined  to  be 
shortly  expelled  from  the  cell ;  or  they  may  be  elements  that 
form  indeed  an  integral  part  of  the  living  texture  of  the  cell 
but  have  been  injuriously  affected  by  the  colouring  matter, 
and  for  that  or  some  other  reason  are  in  a  state  of  diminished 
vitality, — they  are  parts  of  the  cell  that  are  being  killed  by 
the  colouring  reagent  or  that  have  been  totally  killed  by  it, 
whilst  the  rest  survives ;  in  no  case  do  they  consist  of  matter 


158  CHAPTER  XI. 

that  is  fully  and  perfectly  alive.  I  am  inclined  to  think 
that  the  chief  scientific  value  of  the  so-called  vital  or  ivtra- 
vitam  stains  may  be  found  to  lie  in  the  fact  that  they  may 
furnish  us  with  the  means  of  distinguishing  the  living  con- 
stituents of  a  cell  from  the  non-living  ones,  and  even  of 
recognising  amongst  the  living  ones  those  that  possess  only 
a  relatively  low  or  impaired  degree  of  vitality.  See  011  this 
point  (as  on  others  connected  with  the  theory  of  staining) 
the  work  of  FISCHER,  quoted  §  200  A. 

Apart,  however,  from  the  question  whether  the  elements  \ 
stained  by  the  so-called  "  vital "  stains  are  truly  living  or 
not,  it  must  be  conceded  that  this  mode  of  treating  living 
cells  has  frequently  a  considerable  measure  of  practical 
utility.  It  often  enables  us  to  map  out  physiological  or 
morphological  tracts  that  would  otherwise  be  unrecognisable 
or  less  readily  recognisable  in  the  living  state. 

1  find  methyleii  blue,  Congo  red,  neutral  red,  gentian  and 
dahlia  added  to  indifferent  liquids,  extremely  useful  in  the 
examination  of  tissue-cells.  Quinolem  and  Bismarck  brown 
are  well-known  aids  to  the  study  of  Infusoria.  Methylen 
blue  has  a  specific  affinity  for  sensory  nerves,  and  is  an 
extremely  important  reagent  (see  post,  Chap.  XVII) .  Accord- 
ing to  my  experience,  methyleii  blue  is  the  most  generally 
useful  of  these  stains.  It  has  (with  Bismarck  brown,  Congo 
red  and  neutral  red)  the  valuable  point  that  it  is  sufficiently 
soluble  in  saline  solutions,  and  may  therefore  be  employed 
with  marine  organisms  by  simply  adding  it  to  sea  water.  The 
others  are  not  thus  soluble  to  a  practical  extent,  but  I  find 
that  gentian  and  dahlia  become  so  if  a  trace  of  chloral  hydrate 
— 0'25  per  cent,  is  ample  enough — be  added  to  the  saline 
solution.  Any  of  these  reagents  may  be  rubbed  up  with 
serum,  or  other  "  indifferent  "  liquid. 

Methyleii  blue  may  be  fixed  in  the  tissues,  and  permanent 
preparations  made,  by  one  or  other  of  the  methods  described 
in  Chap.  XVII.  Bismarck  brown  stains  may  be  fixed  with  0'2 
per  cent,  chromic  acid  or  with  sublimate  solution  (MAYER), 
or  1  per  cent,  osmic  acid  (LoiSEL,  Journ.  de  VAnat.  et  de  la 
Phys.,  1898,  No.  2,  p.  212 — a  work  that  contains  a  good  deal 
of  information  on  the  subject  of  intra-vitam  stains),  and  the 
preparations  may  be  stained  with  safranin,  care  being  taken 
not  to  expose  them  too  long  to  the  action  of  alcohol. 


STAINING.  159 

202.  Substantive  and  Adjective  Staining ;  Mordants.— In  the 
industry  of  dyeing,  colouring  matters  are  divided  into  two 
classes,  according  to  their  behaviour  with  respect  to  the 
material  to  be  dyed.  Certain  dyes  are  absorbed  directly  from 
their  solution  by  the  material  immersed  therein,  and  combine 
with  it  directly.  In  this  case  the  material  is  said  to  be  tfith- 
tttantively  dyed,  and  the  colouring  matter  is  called  a  substantive 
colouring  matter. 

Other  dyes  do  not  combine  directly  with  the  material  to  be 
acted  on,  but  this  material  must  first  be  charged  with  some 
substance  known  as  mordant  (generally  a  metallic  salt  or 
hydrate)  before  it  will  combine  with  the  colouring  matter. 
These  are  known  as  adjective  colouring  matters.* 

Animal  tissues  have  in  general  a  considerable  affinity  for 
colouring  matters,  taking  them  up  directly  from  their  solutions. 
In  consequence,  the  majority  of  histological  stains  are  obtained 
by  substantive  staining  of  the  tissues.  Still,  as  has  been 
already  pointed  out,  it  seems  probable  that  many  of  the 
histological  stains  that  are  obtained  without  intentional 
mordanting  of  the  tissues,  should  yet  in  strictness  be 
attributed  to  the  class  of  adjective  stains.  This  would  be 
the  case  whenever  there  is  reason  to  suppose  that  the  stain 
obtained  results  from  a  combination  of  the  colouring  matter 
with  some  metallic  salt  or  hydrate  that  is  not  a  constituent 
of  the  living  tissue,  but  has  been  brought  into  it  by  the  fixing 
or  hardening  reagents,  these  reagents  playing  the  part  of 
mordants  though  only  intentionally  employed  for  another 
purpose.  This  would  appear  to  be  the  case  with  the  stains, 
or  some  of  them,  obtained  after  fixation  with  corrosive 
sublimate,  alum,  salts  of  iron,  of  platinum,  of  palladium,  of 
uranium,  and,  for  certain  tissue  elements  and  certain  colours, 
chromium.  And  further,  the  mordanting  substance  may  not 
only  be  present  unintentionally  in  the  fixing  or  hardening 
agents,  it  may  be  present  unintentionally,  or  with  imperfect 
realisation  of  its  import,  in  the  staining  solutions  themselves. 
Such  is  presumably  the  part  played  by  alum  in  many  of  the 
>tains  in  which  it  figures  as  an  ingredient.  Iodine  also 
plays  in  some  staining  processes  a  part  which  seems  only 
explicable  on  the  supposition  that  it  acts  as  a  mordant. 

*  For  an  excellent  popular  exposition  of  this  subject  see  BENEDIKT  and 
KM  IHT'S  'Chemistry  of  the  Coal-tar  Colours'  (George  Bell  and  Sons). 


160  CHAPTER   XI. 

In  some  staining  processes,  however,  mordants  are  inten- 
tionally resorted  to  in  order  to  fix  the  stain.  Mordanting  has 
long  been  employed  in  some  haematein  staining  processes,  such 
as  the  iron-alum  process  of  BENDA  and  M.  HEIDENHAIN.  More 
lately  it  has  been  resorted  to  for  staining  with  tar  colours,  as 
in  the  curious  t{  inversion  "  process  of  RAWITZ.  It  must  be 
admitted  that  mordants  are  in  some  cases  of  use  by  enabling 
us  to  fix  colouring  matter  in  tissue  elements  that  Avould 
otherwise  be  rebellious  to  staining.  And  they  have  in  some 
cases  the  advantage  of  affording  a  very  convenient  means  of 
regressive  staining.  For  it  happens  that  the  colour- com- 
pounds thrown  down  in  mordanted  tissues  are  in  many  cases 
specially  soluble  in  an  excess  of  the  mordant ;  so  that  the 
solution  of  the  mordant  itself  forms  a  very  appropriate 
decolourising  agent. 

Recognising  these  advantages,  it  must  still,  I  think,  be  said 
that  there  seems  to  be  some  danger  at  the  present  moment 
that  the  practice  of  employing  mordants  may  degenerate  into 
an  abuse.  For  surely  the  primary  use  and  intention  of  an 
histological  stain  (not  of  an  industrial  dye)  is,  that  it  should 
select  and  r.eveal  those  elements  of  tissues  that  have  a  natural 
affinity  for  its  colouring  matter.  That  end  is  attained  in  the 
manner  least  open  to  objection  by  the  use  of  substantive 
stains,  the  natural  affinities  of  the  tissues  and  the  colouring 
matter  here  coming  spontaneously  and  unconstrained  into 
play.  Not  so  in  the  case  of  adjective  staining.  Here  the 
colour  is,  as  it  were,  forcibly  compelled  into  an  unnatural  union 
with  all  or  many  of  the  elements  of  the  tissue,  including  many 
which  have  no  natural  affinity  whatever  for  the  colour.  In 
such  preparations  (e.  g.  in  the  "  inversion  "  stain  of  RAWITZ) 
the  distinction  between  chromatic  and  achromatic  elements 
is  obliterated ;  and  the  interpretation  of  the  images  afforded 
by  them  is  open  to  more  serious  causes  of  error  than  in  the 
case  of  substantive  stains. 

The  following  substances  may  be  found  to  act  usefully 
after  the  manner  of  mordants,  for  enhancing*  the  resistance 
of  many  tar  colours  to  the  alcohol  employed  for  decolourising, 
and  for  producing  a  stronger  stain. 

Iodine  :  sections  may  be  treated  for  a  few  minutes  before 
staining  with  tincture  of  iodine. 

Permanganate  of  potash  :  see  HENNEGUY'S  process. 


STAINING.  161 

Formaldehyde  :  see  OHLMACHER'S  process. 

203.  Choice  of  a  Stain. — The  following  may  be  recommended 
to    the   beginner  for  general  work  : — For  sections,  MAYER'S 
h&malum ;    or,    for   chromosmium   objects    more  especially, 
BENDA'S  or  HEIDEN  MAIN'S  iron  haematoxylin. 

For  staining  in  toto  Grenadier's  alcoholic  borax-carmine, 
or  Mayer's  carmalum,  or  haemalum,  unless  the  object  be  so 
impermeable  as  to  require  a  more  highly  alcoholised  stain,  in 
which  case  take  Mayer's  par acar mine,  or  for  chromic  acid 
objects  Mayer's  hasmacalcium. 

For  fresh  tissues  or  small  entire  objects,  methyl  green,  if  it 
is  not  important  to  have  permanent  preparations;  if  it  is, 
take  carmalum  or  alum-carmine  (but  both  of  these  may  give 
precipitates  with  marine  animals). 

Picric  acid  may  be  used  for  double-staining  in  bulk  after 
carmine  or  haematoxylin. 

The  beginner  will  probably  do  well  not  to  use  a  double  stain 
where  a  single  one  will  do.  To  do  so  is  too  often  to  go  farther 
and  fare  worse. 

204.  Staining  Reagents  and  Chemicals. — You  are  not  likely 
to  succeed  in  staining,  especially  in    staining  with   coal-tar 
colours,  unless  you   see    to   it    that   you   are   working  with 
chemicals  of  the  proper  quality.      You  cannot  ensure  this  by 
going   to   a    generally  trustworthy  house  for   chemical    pro- 
ducts— at  all  events,  not  in  the  case  of  coal-tar  colours.      It 
is  not  sufficient   that   these   should   be  what   they  are  com- 
mercially described  to  be ;  they  may  be  pure,  and  yet  not 
give  good  stains.      They  must  (in  the  case  of  coal-tar  colours, 
at  all  events)  be  the  identical  products  used  in  their  work  by 
the  authors  who  have  described  and  recommended  them  (see 
the  note  on  the  numerous  safranins  in  the  market,  sub  voce 
Safranin) .      I  therefore  feel  constrained  to  advise  everybody 
to  get  his  reagents — at  all  events  his  anilins — from  the  well- 
known  chemists  GRUBLER  &  HOLLBORN  or  MUNDER.     Griibler 
&  Hollborn  have  all  the  tried  reagents  in  stock,  and  supply 
only  such  as  have  been  found  by  experiment  with  tissues  to 
furnish   the   desired  stain.      They  also  make  up  fixing  and 
staining    solutions,   injection    and    imbedding    masses,    etc., 
according  to  the  classical  formulae,  and  send  them  out  neatly 

11 


162  CHAPTER   XI. 

packed  and  ready  for  use.  From  experience  I  can  most 
highly  recommend  these  preparations,  which  are  in  nine  cases 
out  of  ten  better  than  those  the  observer  is  likely  to  make  for 
himself.  They  may  be  ordered  from  the  price  list,  or  by 
quoting  the  numbers  of  the  formulae  in  this  work.  The 
address  is  :  Herrn  Dr.  G.  GRUBLER  &  HOLLBORN,  Chemiker, 
Baiersche  Strasse  63,  Leipzig.  They  can  correspond  in 
English. 

Their  preparations  can  be  obtained  in  London  from  Mr. 
CHARLES  BAKER,  244,  High  Holborn,  W.C.,  who  is  also  agent 
for  the  microscopes  and  apparatus  of  Zeiss,  also  for  the 
microtomes  of  Jung,  Becker  and  others,  and  the  bacterio- 
logical apparatus  of  F.  and  M.  Lautenschlaeger,  etc. 

Miinder's  address  is  :  Herrn  Dr.  G.  MUNDER,  Mikroskopisch- 
chemisches  Institut,  Gottingen. 


CHAPTER  XII. 
CARMINE  AND  COCHINEAL  STAINS. 

205.  The  Theory  of  Carmine  Staining, — I  take  the  following 
from  the  important  paper  of  MAYER,  "Ueber  das  Farben 
mit  Carmin,  Cochenille,  und  Hamatein-Thonerde/'  in  Mitth. 
a.  d.  Zool  Station  zu  Neapel,  Bd.  x,  Heft  3,  1892,  p.  480. 
The  rationale  of  staining  with  carmine  has  hitherto  been 
obscured  by  the  erroneous  notion  that  carmine  is  nothing  but 
carminic  acid  with  at  most  certain  impurities.  This  is  not 
the  case.  According  to  the  analysis  of  LIEBEEMANN  (Ber.  d. 
Chem.  Ges.,  Jahrg.  18,  1886,  pp.  1969—1975)  carmine  is  a 
very  peculiar  alumina-hme-protein  compound  of  carminic  acid, 
a  true  chemical  compound  from  which  at  all  events  aluminium 
and  calcium  can  no  more  be  absent  than  sodium  from  salt. 
Analysis  gave  him  about  17  per  cent,  of  water,  20  per  cent, 
nitrogenous  matters,  56  per  cent,  carminic  acid,  at  least  '6 
per  cent,  alumina,  and  3  per  cent,  lime,  together  with  a  small 
proportion  of  magnesia,  potash,  soda,  phosphoric  acid,  and  a 
trace  of  wax.  Mayer  has  come  to  the  conclusion  that  in  the 
processes  of  histological  staining  (not  of  industrial  dyeing) 
the  active  factors  of  the  compound  are,  besides  the  carminic 
acid,  always  the  alumina,  and  in  some  cases  the  lime.  The 
other  bases  are  inactive ;  the  nitrogenous  matters,  so  far  as 
they  have  any  influence  at  all,  are  an  obstacle,  as  it  is  they 
that  give  rise  to  the  well-known  putrefaction  of  the  solu- 
tions. 

Having  arrived  at  these  conclusions,  it  seemed  logical  to 
admit  that  carminic  acid,  instead  of  carmine,  should  be  taken 
a*  the  basis  of  staining  solutions.  This  had  already  been  pro- 
posed by  DIMMOCK,  whose  paper  (Amer.  Natural.,  xviii,  1884, 
pp.  324—7)  I  quoted  at  length  in  the  first  edition  of  this  work. 
But  Dimmock's  proposals  were  not  very  successful,  for  the 


164  CHAPTER    XII. 

reason  that  lie  had  omitted  from  his  solutions  the  essential 
element,  the  alumina.  He  stained,  for  instance,  with  pure 
alcoholic  solution  of  carminic  acid,  or  of  carminate  of 
ammonia.  Such  solutions  stain,  but  stain  weakly  and 
diffusely. 

MAYER  therefore  sought  for  appropriate  means  of  introduc- 
ing the  necessary  alumina  into  the  solutions ;  with  the  results 
that  will  be  set  forth  in  the  next  §. 

206.  Carminic  Acid  occurs  as  a  purple-brown  mass,  easily 
soluble  both  in  water  and  in  alcohol.  It  ought  not  to  be 
hygroscopic,  nor  leave  an  ash  after  glowing  on  a  platinum 
foil.  It  is  (according  to  NTETZKI,  Chemie  der  organischen 
Farbsto/e,  Berlin,  1889,  pp.  231 — 234)  a  weak  (LIEBERMANN 
says  a  strong)  dibasic  acid,  which  forms  soluble  salts  with 
the  alkaline  metals,  insoluble  violet-coloured  ones  with  the 
earthy  and  heavy  metals.  Very  little  is  known  concerning 
the  chemical  nature  of  these  salts. 

The  alumina  salt  (carminate  of  alumina)  has  the  remark- 
able property  of  being  soluble  not  only  in  acids  and  acid  salts, 
such  as  alum,  but  also  in  alkalies  and  alkaline  salts,  such  as 
borax,  provided  that  only  water  or  weak  alcohol  be  employed 
as  the  menstruum.  It  may  be  obtained  by  precipitating  a 
solution  of  carminic  acid  or  of  carminate  of  ammonia  by  means 
of  acetate  of  alumina.  It  is  also  precipitated  from  the  above- 
named  solutions  by  chloride  of  aluminium,  but  only  in  part ; 
whilst  if  alum  be  taken  no  precipitate  is  produced,  the  car- 
minate of  alumina  remaining  in  solution,  and  forming  the 
staining  fluid  given  below  under  the  name  of  Carmalum. 

When  chloride  of  aluminium  is  taken,  a  precipitate  is 
formed,  as  stated  above.  But  this  precipitate  will  redissolve 
if  more  chloride  of  aluminium  be  cautiously  added.  This 
gives  the  staining  fluid  described  in  §  212,  which  may  be 
convenient  in  cases  in  which  it  is  not  desirable  to  work  with 
a  fluid  containing  alum. 

Both  of  these  solutions  stain  in  a  violet  tone,  something 
like  alum-carmine.  A  redder  tone  may  be  obtained  by 
adding  calcium  chloride  to  the  carmalum  solution.  But  this 
is  not  advisable,  for  calcium  chloride  added  to  carmalum 
precipitates  the  solution  with  formation  of  gypsum.  Of 
course,  this  does  not  occur  with  the  aluminium  chloride 


CARMINE    AND   COCHINEAL    STAINS.  165 

<«»liition;  but  for  other  reasons  the  addition  does  not  give 
satisfactory  results  with  the  chloride  of  aluminium  solution 
mentioned  above.  But  it  does  give  good  results  when 
combined  with  an  alcoholic  chloride  of  aluminium  solution, 
and  thus  solves  at  once  the  problem  of  obtaining  a  red  stain 
and  an  alcoholic  staining  fluid.  This  is  described  below 
under  the  name  of  Paracarmine. 

If  the  foregoing  explanations  of  the  rationale  of  carmine 
staining  be  compared  with  the  remarks  on  the  theory  of 
staining  with  haematoxylin  given  in  the  next  Chapter,  an 
interesting  parallelism  will  be  observed.  In  both  processes 
it  is  not  the  colouring  matter  alone  which  is  active,  but  the 
colouring  matter  combined  with  alumina.  The  stain  is 
always  got  with  carminic  acid  +  alumina,  or  with  haematein 
+  alumina ;  other  substances,  such  as  lime,  occasionally 
playing  a  part. 

207.  The  Theory  of  Staining  with  Cochineal. — According  to 
MAYEK,  whose  earliest  researches  are  confirmed  by  his  latest 
Mitth.  Zool.  Stat.  zu  Neapel,  x,  3,  1892,  p.  496),  the  active 
principle   of   extract   or  tincture   of  cochineal    (as   used   in 
histology)    is   not    free    carminic    acid,    but    carminic    acid 
chemically  combined  with  a  base  which  is  not  lime,  but  some 
alkali.      The  pure  aqueous  extract   contains  only  traces  of 
lime,  the  alcoholic  none  at  all.      The  watery  extract   made 
with    alum,    or   cochineal-alum   carmine    (§    214)*   owes    its 
staining    power  to  the  formation  of  carminate  of    alumina 
(for  which  see  last  §).      The  tincture  made  with  pure  alcohol, 
on  the  other  hand,  contains  only  the  above-mentioned  car- 
minate of  some  alkali.       This  carminate  alone  stains  weakly 
and   diffusely    (like    carminic    acid   alone).      But  if   in  the 
tissues  treated  with  it  it  meet  with  lime  salts,  alumina  or 
magnesia  salts,  or  even  metallic  salts  capable  of  combining 
with  it  and  forming  insoluble  coloured   precipitates   in  the 
tis>ues,  then  a  strong  and  selective  stain  may  result.      As  a 
matter  of  fact,  the  simple  cochineal  tincture  of  Mayer  given 
in  §  230  does  give  splendid  results  with  certain  objects  (i.  e. 
such  as  contain  the  salts  in  question).      But  it  is  unfortu- 
nately equally  certain  that  such  objects  are  rather  rare  than 
otherwise,  and  that  with  the  majority  of  objects  the  stain  is 
a  very  poor  one. 


166  CHAPTER   XII. 

But  if  the  necessary  salts  be  added  to  the  tincture  itself, 
then  a  solution  ought  to  result  containing  the  necessary 
elements  for  affording  a  strong  and  selective  stain  with  all 
classes  of  objects.  This  proves  to  be  the  case ;  whence 
Mayer's  new  formula,  §  231. 

208.  General  Remarks. — What  are  the  carmine  stains  use- 
ful for  ?      Is  it  for  staining  fresh  tissues  ?      With  the  excep- 
tion   of    aceto-carmine,    no.      Is    it    for    staining    sections  ? 
Again,  no  ;  for,  in  nine  cases  out  of  ten,  sections  are  better 
stained  by  some  of  the  anilin  stains  and  by  some  hgematein 
stains  than  they  can  be    in   any  carmine   stain.      Is   it   for 
staining  entire  objects  ? — for  staining  in  the  mass  ?      Yes  ; 
for  in  many,  if  not  in  most  cases,  that  can  be   done  more 
satisfactorily  by  means  of  carmine  than  by  means    of   any 
other  known  agent.      For   most  ha3matein  solutions  have  a 
disastrous   tendency  to    overstain ;   and   the   tar- colours    are 
with  hardly  an  exception  entirely  inapplicable  to  staining  in 
bulk. 

Overstains  may  in  all  cases  be  washed  out  with  weak  HC1 
(e..g>  0*1  per  cent.).  HENNEGUY  (Journ.  de  I'Anat.  et  de  la 
Physiol.,  xxvii,  1891,  p.  400)  states  that  overstains  may  be 
completely  removed  by  means  of  permanganate  of  potash. 
(But  that  removes  the  stain  bodily,  rather  than  differentiates 
it.)  All  carmine  stains,  with  the  exception  of  aceto- carmine, 
are  permanent  in  balsam.  None  of  the  acid  stains,  nor  any 
of  Grenadier's  fluids,  should  be  used  with  calcareous 
structures  that  it  is  wished  to  preserve,  unless  they  be  taken 
in  a  state  of  extreme  dilution. 

209.  Choice  of  a  Carmine  Stain. — Grenadier's  alcoholic  borax- 
carmine  may  be  recommended  to  the  beginner  as  being  the 
easiest  of  these  stains  to  work  with.      Carmalum,  or  one  of 
the  alum-carmines,  is  also  an  easy  and  safe  reagent. 

210.  Pure  Carminic  Acid  is  best   obtained  at  present  from 
GRUBLER    &    HOLBORN.      The    price    at    present   is   3s.    per 
10  grms. 

Dr.  MAYER  writes  me  that  samples  obtained  elsewhere  are 
sometimes  not  all  that  could  be  desired,  containing  a  per- 


CARMINE    AND    COCHINEAL    STAINS.  167 

quantity  of  inorganic   impurities,   or   being   hygro- 
scopic, or  being  too  dear. 


A.    AQUEOUS  CARMINE  STAINS. 
a.  Acid. 

211.  MAYER'S  Carmalum  (Mitth.  Zool.  Stat.  zu  Neapel,  x, 
3,  1892,  p.  489). — Carminic  acid,  1  grm. ;  alum,  10  grms. ; 
distilled  water,  200  c.c.  Dissolve  with  heat  (if  necessary  : 
I  have  been  able  to  make  my  solutions  in  the  cold) .  Decant 
or  filter.  Add  some  antiseptic,  either  a  few  crystals  of 
thymol,  or  0*1  per  cent,  salicylic  acid,  or  0'5  per  cent,  salicy- 
late  of  soda.  The  solution  mil  then  keep.  A  clearish  red 
fluid  with  a  violet  tinge.  It  stains  well  in  bulk  even  osmium 
objects.  If  washed  out  with  distilled  water  only,  the  plasma 
will  remain  somewhat  stained.  If  this  be  not  desired,  wash 
out  carefully  with  alum  solution,  or,  in  difficult  cases,  with 
weak  acid,  followed  in  either  case  with  water.  The  general 
effect  is  that  of  an  alum-carmine  stain.  A  notable  difference 
between  the  two  is  that  carmalum  stains  well  in  bulk,  which 
alum -carmine  is  not  very  suitable  for  when  used  in  the 
ordinary  way  ;  but  see  §  215. 

A  weaker  solution  may  be  made  by  taking  from  three  to  five  times  as 
much  alum  and  five  times  as  much  water,  and  dissolving  in  the  cold,  which 
may  be  convenient.  This  is  a  very  close  equivalent  of  alum-carmine, 
giving,  however,  a  somewhat  redder  stain.  I  find  this  solution  very  weak 
for  ordinary  work. 

With  either  solution  the  objects  to  be  stained  should  not 
have  an  alkaline  reaction.  The  other  properties  of  these 
solutions  are  very  similar  to  those  of  alum-carmine. 

UAWITZ  (Anat.  Am.,  xv,  1899,  p.  438)  takes  2  grms.  carminic  acid,  20 
grms.  ammonia-alum,  150  c.c.  water,  and  150  c.c.  glycerin.  A  strongly 
staining  solution  which  keeps  well.  He  recommends  it  only  for  sections. 
Mayer  does  not  admit  the  supposed  advantages  of  the  ammonia-alum. 

212.  MAYER'S  Aqueous  Aluminium-Chloride-Solution  (Mitth.  Zool. 
Stat.  zu  Neapel,  x,  3,  1892,  p.  490). — Carminic  acid  1  grm.,  chloride  of 
aluminium,  3  grms.;  water  200 c.c.  Add  an  antiseptic,  as  for  carmalum. 

CM.-  as  carmalum.  The  stain  is  of  a  blue-violet  colour,  very  powerful, 
and  elective.  But  it  is  not  so  pure  a  stain  as  that  of  carmalum,  plasma 


168  CHAPTER   XII. 

being  more  strongly  coloured.  It  is  recommended  only  as  a  substitute  for 
carmalum  in  cases  in  which  the  latter  is  counter-indicated  on  account  of  the 
presence  of  alum  or  the  like. 

213.  Alum -car  mine  (GRENACHEK'S  formula,  Arch.  mik.  Anat., 
xvi,  1879;  p.  465). — An  aqueous  solution  (of  1  to  5  per  cent, 
strength,  or  any  other  strength  that  may  be  preferred)  of 
common  or  ammonia  alum  is  boiled  for  ten  or  twenty 
minutes  with  ^  to  1  per  cent,  of  powdered  carmine.  (It  is 
perhaps  the  safer  plan  to  take  the  alum  solution  highly  con- 
centrated in  the  first  instance,  and  after  boiling  the  carmine 
in  it  dilute  to  the  desired  strength.)  When  cool  filter. 

This  stain  must  be  avoided  in  the  case  of  calcareous  struc- 
tures that  it  is  wished  to  preserve. 

TIZZONI  (Bull  Sc.  Med.  Bologna,  1884,  p.  259),  PISENTI  (Gazz.  deyli 
Ospetali,  No.  24 ;  Zeit.  f.  wiss.  Mik.,  ii,  1885,  p.  378),  and  GBIEB  (Mem. 
Soc.  Ital.  Sci.,  t.  vi,  No.  9,  1887;  Zeit.  /.  wiss.  Mik.,  vii,  1,  1890,  p.  47) 
have  given  modifications  of  Grenadier's  formula  which  do  not  appear  to  me 
rational. 

MAYEE  (ibid.,  xiv,  1897.  p.  29)  makes  a  stronger  stain  by  taking  2  grms. 
carmine,  5  grms.  alum,  and  100  c.c.  water,  and  boiling  for  an  hour,  which 
sets  some  carminic  acid  free.  The  same  result  may  be  obtained  by  adding 
carminic  acid  to  alum-carmine  or  carmalum. 

Alum-carmine  is  one  of  the  best  stains  to  be  found  outside 
the  coal-tar  colours.  It  is  particularly  to  be  recommended 
to  the  beginner,  as  it  is  easy  to  work  with ;  it  is  hardly 
possible  to  overstain  with  it  (except  muscle).  Its  chief  defect 
is  that  it  is  not  very  penetrating,  and  therefore  quite 
unsuitable  for  staining  objects  of  considerable  size  in  bulk. 
This  defect  may,  however,  be  to  some  extent  overcome  by 
employing  the  acid  formula  of  Henneguy  (§  215),  if  it  be  not 
convenient  to  use  Mayer's  carmalum. 

The  stain  is  permanent  in  balsam ;  as  to  aqueous  media  I 
cannot  say. 

214.  Cochineal  Alum-carmine  (PARTSCH,  Arch.  f.  mik.  Anat., 
xiv,  1877,  p.  180). — Powdered  cochineal  is 'boiled  for  some 
time  in  a  5  per  cent,  solution  of  alum,  the  decoction  filtered^ 
and  a  little  salicylic  acid  added  to  preserve  it  from  mould. 

Another  method  of  preparation  has  been  given  by  CZOKOE  (Arch.f.  mik 
Anat.,  xviii,  1880,  p.  413).  Mayer  has  carefully  examined  both,  and  find? 


CARMINE    AND    COCHINEAL    STAINS.  169 

that  Partsch's  is  the  more  rational,  the  proportion  of  alum  in  it  being 
exactly  right,  whilst  in  Czokor's  it  is  insufficient.  Partsch's  fluid  has  also 
the  advantage  of  keeping  better. 

RABL  (Zeit.  f.  wiss.  Mik.,  xi,  2,  1894,  p.  168)  takes  25  grms.  each  of 
cochineal  and  alum,  800  c.c.  of  water,  and  boils  down  to  600  c.c. 

These  solutions  are  to  all  intents  and  purposes  "  alum- 
carmines."  They  give  a  stain  that  is  practically  identical 
with  that  of  alum- carmine  made  from  carmine,  with  perhaps 
even  more  delicate  differentiations  (but  that  depends  so  much 
on  the  quality  of  the  carmine,  the  quality  of  the  cochineal, 
and  the  nature  of  the  objects  to  be  stained,  that  no  absolute 
rule  can  be  stated).  They  should  be  used  in  exactly  the 
same  way  as  the  carmine  fluid. 

215.  Acetic   Acid   Alum- Carmine  (HENNEGUY,  in   Traite  des 
M'-th.  techn.,  LEE  et  HENNEGUY,  1887,  p.  88). — Excess  of  car- 
mine is  boiled  in  saturated  solution  of  potash  alum.      After 
cooling  add  10  per  cent,  of  glacial  acetic  acid,  and  leave  to 
settle  for  some  days,  then  filter. 

For  staining,  enough  of  the  solution  is  added  to  distilled 
water  to  give  it  a  deep  rose  tint.  In  order  to  ensure  rapid 
diffusion,  it  is  well  to  bring  the  tissues  into  the  stain  direct 
from  alcohol.  Stain  for  twenty -four  to  forty-eight  hours, 
and  wash  for  an  hour  or  two  in  distilled  water.  Mount  in 
balsam.  You  can  mount  in  glycerin,  but  the  preparations 
do  not  keep  so  well  as  in  balsam. 

The  advantage  of  this  carmine  is  that  it  has  greater  power 
of  penetration  than  the  non-acidified  alum-carmine,  and  stains 
deep-seated  layers  of  tissue  just  as  well  as  the  superficial 
ones.  The  colour  of  the  stain  is  a  somewhat  inelegant  violet, 
but  this  can  be  changed  to  a  warmer  tone  by  treating  the 
objects  with  dilute  HC1,  as  for  borax-carmine  objects. 

216.  Alum-Carmine  and  Picric  Acid. — Alum-carmine  objects 
may    be    double-stained   with   picric   acid.      LEGAL   (Morph. 
Jahrb..  viii,  p.  353)  combines  the  two  stains  by  mixing  ten 
vols.  of  alum-carmine  with  one  of  saturated  picric  acid  solu- 
tion.     I  consider  this  to  be  a  very  recommendable  practice. 

217.  Aceto-Carmine  (Acetic  Acid  Carmine)  SCHNEIDEE'S  formula, 
Zool.  Anzeig.,  No.  56,  1880,  p.  254). — To  boiling  acetic  acid  of  45  per  cent. 


170  CHAPTER   XII. 

strength  add  carmine  until  no  more  will  dissolve  and  filter.  (Forty-five  per 
cent,  acetic  acid  is,  according  to  Schneider,  the  strength  that  dissolves  the 
largest  proportion  of  carmine.) 

To  use  the  solution  you  may  either  dilute  it  to  1  per  cent,  strength,  and 
use  the  dilute  solution  for  slow  staining  ;  or  a  drop  of  the  concentrated 
solution  may  be  added  to  a  fresh  preparation  under  the  cover-glass.  If  you 
use  the  concentrated  solution  it  fixes  and  stains  at  the  same  time,  and  hence 
may  render  service  for  the  study  of  fresh  objects.  It  is  very  penetrating, 
a  quality  that  enables  it  to  be  used  where  ordinary  reagents  would  totally 
fail.  The  stain  is  a  pure  nuclear  one.  Unfortunately  the  preparations 
cannot  be  preserved,  and  for  this  and  other  reasons  the  stain  is  of  very 
restricted  applicability. 

A  similar  stain  has  been  prepared  with  formic  acid  by  PIANESE  (see  Zeit. 
f.  wiss.  Mik.,  x,  4,  1894,  p.  502).  Probably  for  almost  all  the  purposes  for 
which  aceto-carmine  is  useful,  methyl-green  will  give  better  results. 

For  BURCHARDT'S  pyroligneous-acid  carmines  see  Arch.  f.  mik.  Anat.,  liii, 
1898,  p.  232  :  Zeit.f.  wiss.  Mik.,  xv,  4,  p.  453  ;  Journ.  Roy.  Mic.  Soc.,  1899, 
p.  453. 

218.  Iron  Carmine  (ZACHARIAS,  Zool.  Anz.,  No.  440,  1894, 
p.  62). — Stain  for  several  hours  in  carmine  (Zacharias  stains 
in  an  aceto-carmine,  of  which  I  suppress  the  formula,  for,  as 
pointed  out  to  me  by  Dr.  MAYER,  and  as  I  have  verified, 
carmalum  does  just  as  well) .  Rinse  the  objects  with  dilute 
acetic  acid,  and  bring  them  (taking  care  not  to  touch  them 
with  metallic  instruments  if  the  aceto-carmine  have  been 
taken)  into  a  1  per  cent,  solution  of  ammoniated  citrate  of 
iron  (the  pharmaceutical  Ferri  et  Ammonias  Citras}.  Leave 
them  till  thoroughly  penetrated,  for  as  much  as  two  or  three 
hours  if  need  be.  In  this  solution  they  take  on  a  black  tint 
(with  sections  this  happens  in  a  few  minutes).  They  should 
be  removed  as  soon  as  the  reaction  has  taken  place  through- 
out, otherwise  there  is  risk  of  over-blackening.  Wash  for 
several  hours  in  distilled  water,  dehydrate  and  mount  in 
balsam. 

This  is  at  the  same  time  a  chromatin  stain  and  a  plasma 
stain.  In  my  preparations  chromatin  is  blue  and  plasmatic 
elements  brown.  I  consider  the  method  may  render  service 
in  some  cases. 

PFEIFFER  VON  WELLHEIM  (Zeit.  f.  wiss.  Mik.,  xv,  1,  1898,  p.  123) 
mordants  for  six  to  twelve  hours  in  a  very  weak  solution  of  chloride  of 
iron  in  50  per  cent,  alcohol,  washes  in  50  per  cent,  alcohol,  and  stains  for  a 
few  hours  in  a  dilute  solution  of  carminic  acid  in  50  per  cent,  alcohol. 
Overstains  may  be  corrected  with  O'l  to  0'5  per  cent.  HC1  alcohol. 


CARMINE   AND    COCHINEAL    STAINS.  171 


)3.  So-called  "  Neutral,"  and  Alkaline. 

219.  Ammonia-Carmine. — In  my  opinion  there  is  no  valid  excuse  for 
using  ammonia-carmine  at  all  at  the  present  day. 

If.  however,  such  a  stain  be  used,  care  should  be  taken  to  get  rid  of  the 
free  ammonia  as  completely  as  possible.  This  may  be  done  by  boiling  until 
the  excess  of  ammonia  has  evaporated.  (So  long  as  free  ammonia  is  present 
large  bubbles  are  formed  in  the  fluid,  and  the  latter  shows  a  dark  purple 
colour.  When  the  free  ammonia  has  evaporated  small  bubbles  appear,  and 
the  solution  takes  a  brighter  red  tint.) 

One  per  cent,  each  of  carmine  and  ammonia  in  distilled  water  is  a  good 
proportion. 

But  a  safer  mode  of  preparation  is  that  of  RANVIER,  as  follows  (kindly 
communicated  by  Dr.  MALASSEZ,  see  Traite  des  Methodes  techniques,  etc., 
of  Lee  and  Henneguy,  1st  edit.,  p.  82). — Make  a  simple  solution  of 
carmine  in  water  with  a  slight  excess  of  ammonia,  and  expose  it  to  the  air 
in  a  deep  crystallising  dish  until  it  is  entirely  dried  up.  It  should  be 
allowed  to  putrefy  if  possible.  Dissolve  the  dry  deposit  in  pure  water,  and 
filter. 


220.  Magnesia-carmine  (MAYER,  Zeit.f.  wiss.  Mik.,  xiv,  1897,  p.  23). 
— Take  1  grm.  carmine,  O'l  grm.  magnesia  usta,  and  50  c.c.  distilled  water, 
boil  for  five  minutes,  filter,  and  add  three  drops  of  forinol.     This  is  the 
stock  solution.     A  weak  solution  may  be  made  by  boiling  O'l  grm.  carmine 
for  half  an  hour  in  50  c.c.  of  magnesia  water  (made  by  leaving  O'l  grm.  of 
magnesia  usta  in  contact  with  100  c.c.  of  spring  water  for  a  week  with 
frequent  agitation,  and  decanting  when  required  for   use).     Mayer   says 
these   solutions   are    less    injurious   to   tissues   than    the    other    alkaline 
carmines. 

221.  As  to  Picro-carmine. — The  term  "  picro-carmine"  is  commonly 
used  to  denote  a  whole  tribe  of  solutions  in  which  carmine,  ammonia,  and 
picric  acid  exist  uncombined  in  haphazard  proportions.     RANYIER,  to  whom 
we  owe  the  invention  of  picro-carmine,  claims  that  when  prepared  by  his 
process  it  results  as  a  definite  chemical  substance,  a  double  salt  of  picric  and 
carrainic  acid  and  ammonia,  or  picrO'Carminate  of  ammonia.     But  this  is 
certainly  not  the  case,  for  carmine  is  not  carrainic  acid,  see  supra,  §  205. 
It  should  be  understood  that  the  raison  d'etre  of  picro-carmine  does  not  lie 
in  its  capacity  of  affording  a  double  stain.     The  double  stain,  if  that  is  all 
that  is  wanted,  can  be  just  as  well  or  better  obtained  by  staining  first  with 
borax-carmine,  or  the  like,  and  after-staining  with  picric  acid.    The  essential 
point  about  picro-carmine  is  that  it  is  less  alkaline  than  ammonia-carmine. 
Ranvier    was,  in   fact,  led  to  add  picric  acid  to  ammoniacal  solution  of 
carmine  by  the  desire  of  neutralising  the  ammonia,  that  is  all.     But  it 
always  is  alkaline,  and  is  frequently  injurious  to  tissues. 

Compare  the  paper  of  MAYER,  Ueber  Pikrocarmin,  in  Zeit.f.  wiss.  Mik., 
xiv,  1,  1897,  p.  18. 


172  CHAPTER   XII. 

222.  KANVIEE'S  Picro-carmine,  Original  Formula  (Traite,  p.  100). 
To  a  saturated  solution  of  picric  acid  add  carmine  (dissolved  in  ammonia)  to 
saturation.     Evaporate  down  to  one  fifth  the  original  volume  in  a  drying 
oven  ;  and  separate  by  filtration  the  precipitate,  pour  in  carmine,  that  forms 
in  the  liquid  when  cool.     Evaporate  the  mother-liquid  to  dryness,  and  you 
will  obtain  the  picro-carmine  in  the  form  of  a  crystalline  powder  of  the 
colour  of  red  ochre.     It  ought  to  dissolve  completely  in  distilled  water  ;  a 
1  per  cent,  solution  is  best  for  use. 

For  slow  staining,  dilute  solutions  may  advantageously  have  1  or  2  per 
cent,  of  chloral  hydrate  added  to  them. 

Overstains  may  be  washed  out  with  hydrochloric  acid,  say  O'o  per  cent.,  in 
water,  alcohol,  or  glycerin. 

Preparations  should  be  mounted  in  balsam,  or  if  in  glycerin,  this  should 
be  acidulated  with  1  per  cent,  of  acetic  acid,  or  better,  formic  acid. 

223.  RANVIEE'S  Newer  Formula  does  not  give  a  more  constant  pro- 
duct (see  previous  editions). 

224.  MAYER'S   Pier o -magnesia   Carmine   (Zeit.  f.   wiss.  Mik.,  xiv, 
1897,  p.  25)  is  relatively  constant  and  innocuous  to  tissues.     It  consists  of 
1  vol.  of  the  stock  solution  of  magnesia-carmine  (§  220),  and  10  vols.  of  a 
0'6  per  cent,  solution  of  picrate  of  magnesia,  or  of  equal  parts  of  the  weak 
solution   and   the    picrate   solution.     The   picrate    may  be   obtained  from 
GEUBLEE   &  HOLLBOBN,  or  the  solution  may  be   made  by  heating  0'25 
grms.  of  carbonate  of  magnesia  in  200  c.c.  of  0'5  per  cent,  solution  of  picric 
acid,  allowing  to  settle  and  filtering. 

225.  Other   Formulae  for   Picro-carmine. — I    have   tried   most  of 
them,  and  found  no  real  advantage  in  any  of  them  (see  previous  editions). 

226.  Other   Aqueous   Carmines    (Acid    and   Alkaline). — For  all 
of  them  see  previous  editions. 


B.  ALCOHOLIC  CARMINE  STAINS. 

227.  Alcoholic  Borax-carmine  (GRENACHER,  Arch.  f.  Mik. 
Aiiat.,  xvi,  1879,  p.  466,  et  seq.). — Make  a  concentrated 
solution  of  carmine  in  borax  solution  (2  to  3  per  cent,  carmine 
to  4  per  cent,  borax)  by  boiling  for  half  an  hour  or  more  ; 
dilute  it  with  about  an  equal  volume  of  70  per  cent,  alcohol, 
allow  it  to  stand  some  time  (twenty-four  hours — MAYER), 
and  filter.  Or  the  mixture  of  carmine  and  l)orax  solution  is 
allowed  to  fitand  for  two  or  three  days  and  occasionally  stirred  ;' 
the  greater  part  of  the  carmine  will  dissolve.  To  the  solution 
is  added  an  equal  bulk  of  70  per  cent,  alcohol ;  the  mixture 


CARMINE   AND   COCHINEAL    STAINS.  173 

to  stand  for  a  week,  and  then  is  filtered.  If  on 
keeping  more  carmine  is  deposited,  it  must  be  refiltered. 

Preparations  should  remain  in  the  stain  until  they  are 
thoroughly  penetrated  (for  days  if  necessary),  and  then  be 
brought  (without first  washing  out)  into  alcohol  (of  70  per  cent. ; 
this  is  absolutely  necessary — MAYER)  acidulated  with  4  to  6 
drops  of  hydrochloric  acid  to  each  100  c.c.  of  alcohol.  They 
are  left  in  this  until  the  stain  is  differentiated,  and  may  then 
be  washed  or  hardened  in  neutral  alcohol.  Four  drops  of 
HC1  is  generally  enough.  Three  drops  I  find  not  quite 
sufficient.  The  stained  objects  should  remain  in  the  acidu- 
lated alcohol  till  they  acquire  a  bright  transparent  look. 
This  may  require  days  (MAYER). 

For  delicate  objects,  and  for  very  impermeable  objects,  it 
may  be  well  to  increase  the  proportion  of  alcohol  in  the 
stain ;  it  may  conveniently  be  raised  to  about  50  per  cent. 
It  should  not  exceed  60  per  cent,  in  any  case  (MAYKK). 

This  stain  is  probably  by  far  the  most  popular  of  any  for 
staining  in  bulk.  It  is  easy  to  use,  and  gives  a  most 
splendid  coloration.  But  it  is  not  so  penetrating  as  is 
commonly  supposed,  and  has  the  defect  of  sometimes  form- 
ing precipitates  in  the  cavities  of  bulky  objects  which  cannot 
be  removed  by  washing  out.  And  it  must  be  remembered 
that  the  fluid  is  alkaline,  and  therefore  may  not  be  suitable 
for  delicate  cytological  work. 


\.  MAYER'S  Paracarmine  (Mitth.  Zool.  Stat.  zu  Neap'el, 
x,  3,  1802,  p.  491). — Carminic  acid,  1  grm. ;  chloride  of 
aluminium,  0'5  grm.  ;  chloride  of  calcium,  4  grins.  ;  70  per 
cent,  alcohol,  100  c.c.  Dissolve  cold  or  warm,  allow  to 
settle,  and  filter.  A  light  red  liquid,  specially  adapted  for 
staining  in  bulk,  and  much  like  Grenadier's  alcoholic  borax- 
carmine. 

Objects  to  be  stained  should  not  have  an  alkaline  reaction, 
nor  contain  any  considerable  amount  of  carbonate  of  lime 
(spicules  or  skeletal,  parts  of  corals,  etc.)  which  would  give 
rise  to  precipitates  (MAYER).  Wash  out  sections,  or  objects 
intended  to  be  sectioned,  with  pure  70  per  cent,  alcohol. 
Objects  intended  to  be  mounted  whole  may  be  washed  out 
with  a  weak  solution  of  aluminium  chloride  in  alcohol,  or  if 
this  be  not  sufficient,  with  5  per  cent,  common  acetic  acid  (or 


174  CHAPTER    XTT. 

2'5  per  cent,  glacial  acetic  acid)  in  alcohol.  This  may  also 
be  done  with  section-material,  if  it  is  desired  to  obtain  a 
more  purely  nuclear  stain. 

For  staining  bulky  objects  with  large  cavities,  such  as 
Salpa,  the  solution  should  be  diluted  (with  alcohol)  ;  and  as 
this  may  cause  precipitates  to  form  during  the  staining, 
especially  if  the  objects  are  hot  very  clean,  it  is  advisable  to 
slightly  acidify  ihe  dilute  solutions. 

Paracarmine  gives  a  nuclear  stain  of  a  red  colour,  though 
not  so  fiery  red  as  that  of  borax -carmine.  Its  points  of 
superiority  over  borax-carmine  are  that  it  is  less  hurtful  to 
tissues ;  that  it  is  more  highly  alcoholic,  therefore  more 
penetrating  ;  that  it  has  less  tendency  to  form  granular  pre- 
cipitates in  the  interior  of  objects,  and  that  it  generally 
keeps  perfectly  without  precipitating  (mine  has  precipitated 
somewhat,  though  not  to  an  injurious  extent). 

229.  Alcoholic  Hydro  chloric- Acid  Carmine. — Sometimes  it  is 
desirable  to  possess  a  powerful  staining  medium  more  highly  alcoholised 
than  the  foregoing,  and  of  acid  reaction.  Hydrochloric-acid  carmine 
possesses  these  qualities,  and  may,  for  instance,  be  frequently,  useful  in 
work  on  Arthropoda,  especially  the  marine  forms. 

GEENACHEE'S  receipt  (Arch.  f.  miJc.  Anat.,  xvi,  1879,  p.  468)  will  be 
found  extremely  troublesome  by  those  who  are  not  expert  at  neutralising. 
The  following  method,  due  to  PAUL  MAYEE  (Mitth.  Zool.  8tat.  Neapel,  iv, 
1883,  p.  521 ;  Journ.  Roy.  Mic.  Soc.  [N.S.],  iv,  1884,  p.  317 ;  Intern. 
Monatsschr.  f.  Anat.,  etc.,  1897,  p.  43),  is  better : — Carmine  4  grms.  ;  water, 
15  c.c. ;  hydrochloric  acid,  30  drops.  Boil  till  the  carmine  is  dissolved,  add 
95  c.c.  of  85  per  cent,  alcohol,  and  neutralise  by  adding  ammonia  until  the 
carmine  begins  to  precipitate. 

If  it  be  desired  to  dilute  the  solution,  it  should  be  done  with  alcohol,  not 
water,  and  alcohol  of  80  to  90  per  cent,  should  be  taken  for  washing  out. 

If  it  be  desired  to  have  a  purely  nuclear  stain,  the  alcohol  must  be  very 
slightly  acidulated  with  HC1. 

230.  Alcoholic  Cochineal,  MAYER'S  Old  Formula  (Mitth.  Zool. 
Stat.  Neap.,  ii,  1881,  p.  14). — Cochineal  in  coarse  powder  is 
macerated  for  several  days  in  alcohol  of  70  per  cent.  For 
each  gramme  of  the  cochineal  there  is  required  8  to  10  c.c. 
of  the  alcohol.  Stir  frequently.  Filter,  and  the  resulting 
clear,  deep  red  solution  is  fit  for  staining.  (If  the  filter 
paper  should  contain  much  lime,  flakes  of  lime  carminate 
may  be  thrown  down.) 

The  objects  to  be  stained  must  previously  be  imbibed  with 


CARMINE    AND    COCHINEAL    STAINS.  175 

alcohol  of  70  per  cent.,  and  alcohol  of  the  same  strength  must 
be  used  for  washing  out  or  for  diluting  the  staining  solution. 
The  washing  out  must  be  repeated  with  fresh  alcohol  until 
the  latter  takes  up  no  more  colour.  Warm  alcohol  acts 
more  rapidly  than  cold.  Overstaining  seldom  happens  ;  it 
may  be  corrected  by  means  of  70  per  ceot  alcohol,  contain- 
ing 3*3-  per  cent,  hydrochloric  or  1  per  cent,  acetic  acid. 

Small  objects  and  thin  sections  may  be  stained  in  a  few 
minutes ;  larger  animals  require  hours  or  days. 

A  nuclear  stain,  slightly  affecting  protoplasm.  The  colour 
varies  with  the  reaction  of  the  tissues,  and  the  presence  or 
absence  of  certain  salts  in  them.  Crustacea  with  thick 
chitinous  integuments  are  generally  stained  red,  most  other 
organisms  blue.  The  stain  is  also  often  of  different  colours 
in  different  tissue  elements  of  the  same  preparation.  Glands 
or  their  secretion  often  stain  grey- green. 

Acids  lighten  the  stain  and  make  it  yellowish  red. 
Caustic  alkalies  turn  it  to  a  deep  purple. 

The  best  stains  are  obtained  in  the  case  of  objects  that 
have  been  prepared  with  chromic  or  picric  acid  combinations, 
or  with  absolute  alcohol.  Osmic  acid  preparations  stain 
very  weakly  unless  they  have  been  previously  bleached.  All 
acids  must  be  carefully  washed  out  before  staining,  or  a 
diffuse  stain  will  result.  The  stain  is  permanent  in  oil  of 
cloves  and  balsam. 

The  object  for  which  this  stain  was  imagined  is  to  obtain 
an  alcoholic  staining  fluid  whose  high  penetrating  pewer 
allows  it  to  be  employed  in  the  case  of  organisms,  such  as 
Arthropoda,  whose  chitinous  investments  are  but  very  slightly 
permeable  by  aqueous  solutions. 

This  fine  stain  has  over  the  new  fluid  (next  §)  the  (for 
some  cases  considerable)  advantage  of  being  more  highly 
alcoholic ;  and  it  does  not  contain  free  acid,  so  that  it  can  be 
used  with  calcareous  structures  which  it  is  wished  to  preserve 
— which  the  new  fluid  cannot.  For  specimens  of  Pluteus, 
for  instance,  I  find  it  excellent. 


231.  MAYEB'S  Alcoholic  Cochineal,  New  Formula  (Mitth.  Zool. 
Stat.  Neapel,  x,  3, 1892,  p.  498). — Cochineal,  5  grms. ;  chloride  of  calcium, 
o  grms.;  chloride  of  aluminium,  O'o  grm. ;  nitric  acid  of  1P20  sp.  gr.,  8 
drops  ;  50  per  cent,  alcohol,  100  c.c.  Powder  the  cochineal  finely  and  rub 


176  CHAPTER  XII. 

up  in  a  mortar  with  the  salts,  add  the  alcohol  and  acid,  heat  to  boiling-point, 
leave  to  cool,  leave  for  some  days  standing  with  frequent  agitation,  filter. 

Use  as  the  old  tincture,  the  objects  being  prepared  and  washed  out  with 
50  per  cent,  alcohol.  The  stain  is  like  that  of  paracarmine,  but  not  quite  so 
strong  and  not  so  sharp.  Mayer  only  recommends  it  as  a  succedaneum,  of 
paracarmine. 

Since  this  fluid  contains  in  itself  all  the  necessary  salts  (see  §  207),  it 
has  over  the  old  one  the  advantage  of  giving  good  results  with  all  classes  of 
objects,  with  the  disadvantage  of  being  less  highly  alcoholic. 


CHAPTER    XIII. 
HAMATE  IN  (H^IMATOXYLIN)  STAINS. 

232.  Theory  of  Haematoxylin  Staining — It  appears  to  be  now 
thoroughly  well  established  (see  NIETZKI,  Chemie  der  organ- 
ischen  F&rb*toffe3  Berlin,  Springer,  1889,  pp.  215 — 217,  and 
MAYER,  Mitth.  Zool.  Stat.  Neapel,  x,  1891,  p.  170)  that  the 
active  colouring  principle  of  the  usual  histological  staining 
solutions  is  hsematein, — a  product  of  the  oxidation  of  their 
contained  haematoxylin  by  means  of  the  air  to  which  they 
are  exposed  (see  MAYER,  "  Ueber  das  Farben  mit  Hamatoxy- 
lin"  in  Mitth.  a.  d.  Zool.  Station  zu  Neupel,  Bd.  x,  Heft  1, 
189J,  pp.  170 — 186;  UNNA,  "  Ueber  die  Reifung  unserer 
Farbsto/e,"  in  Zeit.  f.  u-is*.  Mik.,  viii,  4,  1892,  p.  483).  This 
change  is  known  as  "  ripening,"  and  until  it  has  taken  place 
the  solutions  are  not  fit  to  use  for  staining. 

Hitherto  it  has  been  the  practice  to  rely  (quite  uncon- 
sciously so  far  as  the  chemical  theory  is  concerned)  on  the 
spontaneous  absorption  by  the  solutions  of  oxygen  from  the 
air  to  effect  this  "  ripening/'  but  it  has  now  been  discovered 
(by  both  MAYER  and  UNNA  independently)  that  nothing  is 
easier  than  to  bring  about  the  reaction  artificially  •  all  that 
is  necessary  being,  for  instance,  to  add  to  a  solution  of  haema- 
toxylin containing  alum  a  little  neutralised  solution  of  per- 
oxide of  hydrogen  or  other  powerful  oxidising  agent.  The 
solution  becomes  almost  instantaneously  dark  blue,  "  ripe," 
and  fit  for  staining,  thus  definitely  confirming  the  truth  of 
the  hypothesis. 

A  solution  of  pure,  uncombined  haematein,  however,  would 
not  afford  a  selective  stain  such  as  we  require  in  histology  ; 
it  would  be  at  most  a  feeble  dye.  The  usual  solutions  (I  am 
not  here  speaking  of  Weigert's  or  Heidenhain's  or  similar 
processes)  all  contain  alum,  and  Mayer  holds  that  the  active 

12 


178  CHAPTER  XIII. 

agent  in  them  is  a  compound  of  haematein  with  alumina 
(much  as  in  carmine  the  active  agent  is  a  compound  of  car- 
minic  acid  with  alumina,  cf.  §  206).*  These  solutions  of 
alumina-hgematein  lakes  are  in  general  the  ones  employed 
for  substantive  staining  (see  §  202).  Besides  these,  some 
other  compounds,  viz.  those  of  chrome,  iron,  copper,  vana- 
dium, and  molybdenum,  are  also  employed  in  histology,  but 
mostly  in  the  adjective  way  of  staining  (§  202).  The  com- 
pounds of  haematoxylin  with  the  other  heavy  or  alkaline 
metals  have  been  tried,  but  do  not  afford  histological  stains. 
Neglecting  all  these  for  the  present,  let  us  return  to  the 
consideration  of  the  stains  composed  of  the  alumina  lakes. 

The  tirst  difficulty  with  which  the  worker  with  these 
solutions  has  to  contend  is  that  of  getting  his  haematoxylin 
duly  oxidised  into  haematein,  in  order  to  the  formation  of  the 
desired  hasmatein-alumina  compound,  or  lake.  If  this  be 
done  by  the  hitherto  customary  process  of  leaving  the  solu- 
tions to  "  ripen  "  by  the  action  of  the  air,  it  is  necessary  to 
wait  for  a  long  time  before  the  reaction  is  obtained.  During 
all  this  time,  it  may  be  weeks  or  months,  there  is  no  means, 
except  repeated  trial,  of  ascertaining  whether  the  solution  at 
any  moment  contains  sufficient  hsematein  to  afford  a  good 
stain.  And  here  a  second  difficulty  arises ;  the  oxidising 
process  continuing,  the  solutions  become  (t  over-ripe  ;  "  the 
haematein,  through  further  oxidation,  passes  over  into  colour- 
less compounds,  and  the  solutions  begin  to  precipitate.  They 
are  therefore,  in  reality,  a  mixture  in  constantly  varying 
proportions  of  "  unripe,"  "ripe/'  and  "  over-ripe "  con- 
stituents (the  first  and  last  being  useless  for  staining  pur- 
poses), and,  in  consequence,  their  staining  power  is  very 
inconstant. 

The  great  point  in  Mayer's  work  is  that  not  haematoxylin, 
but  hsematein,  should  be  taken  in  the  first  instance  for  making 
the  staining  solutions.  This  at  once  relieves  us  from  the 
tedious  and  uncertain  process  of  "ripening"  in  the  old  way. 
We  have  a  ripe  solution  to  begin  with,  and  we  know  that  it 
must  be  ripe.  A  discovery  of  Unna's,  to  be  mentioned  below, 

*  I  restrict  this  assertion  to  the  case  of  the  alurmna-haematein  stains,  as 
it  seems  possible  that  in  the  iron  or  chrome  processes  the  colouring  agent 
may  be  haBinatoxylin,  or  even  some  higher  oxidation  product  of  it  than 
hsematein.  Cf.  MAYEE  in  Anat.  Anz.,  xiii,  1897,  p.  318. 


HJCMATEIN  (HJSMATOXYLIN)  STAINS.  179 

nffords  a  means  of  preventing  the  "  over-ripening  "  brought 
about  by  excessive  oxidation. 

If,  however,  it  be  still  preferred  to  use  hsematoxylin,  this 
should  not  be  done  by  dissolving  the  haematoxylin  crystals 
straight  away  in  the  other  ingredients  of  the  solution.  The 
solutions  should  be  made  up  from  a  strong  stock  solution 
made  by  dissolving  haematoxylin  crystals  in  absolute  alcohol  : 
one  in  ten  is  a  good  proportion.  This  solution  should  be 
kept  for  a  long  time — months,  at  least,  a  year  if  possible  ; 
it  gradually  becomes  brown,  and  should  not  be  used  till  it 
has  become  quite  dark.  It  has  then  become  to  a  great  extent 
oxidised  into  haematein,  and  the  staining  solutions  made  up 
from  it  will  be  at  once  fairly  ripe. 

233.  UNNA'S  Half-ripe  Constant  Stock  Solution  (Zeit.f.  wiss.  Mik., 
viii,  4,  1892,  p.  483). — A  ripe  solution  may  be  made  constant  (see  last  §) 
by  simply  adding  a  reducing  agent  to  it.  Various  reducing  agents  are 
available  for  tbis  purpose  (see  tbe  original  paper)  ;  the  most  convenient 
method  is  the  simple  addition  of  a  little  sulphur.  The  following  formula  is 
recommended  : 

Hsematoxylin .         .1 

Alum 10 

Alcohol 100 

Water 200 

Sublimed  sulphur 2 

If  the  sulphur  be  added  to  the  hsBmatoxylin  solution  only  when  the  latter 
has  become  somewhat  strongly  blue,  i-  e.  after  two  or  three  days'  time,  the 
stage  of  oxidation  attained  by  the  solution  will  be  fixed  by  the  sulphur. 
The  solution  in  this  state  may  be  used  for  staining.  MAYER  (Mitth.  Zool. 
Stat.  Neapel,  xii,  2,  1896,  p.  309)  finds  that  the  sulphur  process  does  not 
preserve  the  solutions  for  long,  whilst  for  some  unexplained  reason  the 
simple  addition  of  glycerin  does ;  see  below,  "  GLYCH.EMALUM." 

234.  Concerning  Haematein. — The  following  is  taken  from  the 
two  papers  of  MAYER  quoted  above,  §§  205,  232.  Haematein 
is  entirely,  though  with  difficulty,  soluble  in  distilled  water 
and  in  alcohol,  giving  a  yellowish-brown  solution,  which 
remains  clear  on  addition  of  acetic  acid.  Alkalies  dissolve 
it  with  a  blue-violet  tint. 

It  is  now  found  in  commerce  ;  but  MAYER  has  hitherto  only 
been  able  to  procure  it  in  a  perfectly  pure  state  from  GEIGY 
&  Co.,  in  Bale.  But  there  is  also  found  in  commerce  an 
ammonia-compound  of  haematein — Hsematein-Ammoniak,  also 
known  in  commerce  as  Hsemateinum  crystallisatum  ;  this  may 


180  CHAPTER    XIT1. 

be  obtained  in   a    sufficiently   pure   state    from    GRUBLER   & 
HOLLBORN. 

This  is  somewhat  more  easily  soluble  in  both  water  and 
alcohol  than  hsematein  is,  and  does  quite  as  well  for  staining 
purposes.  The  histologist  can  easily  prepare  it  for  himself 
as  follows  : 

235,  Haemateate  of  Ammonia  (MAYER,  Mitth.  Zool .  Stat. 
Neapel,  x,  1891,  p.  172). — Dissolve  1  grrn.  of  heematoxylin 
with  the  aid  of  heat  in  20  c.c.  of  distilled  water,  filter 
if  necessary,  add  1  c.c.  of  caustic  ammonia  (of  0'875 
sp.  gr.),  and  bring  the  purple  liquid  into  a  capsule  of  such 
dimensions  that  its  bottom  be  not  covered  to  a  depth  of  more 
than  half  a  centimetre.  Let  the  liquid  evaporate  at  the 
ordinary  temperature  and  protected  from  dust.  The  dry 
product  will  consist  of  haemateate  of  ammonia,  about  equal 
in  weight  to  the  hsematoxylin  taken  in  the  first  instance. 
The  evaporation  should  not  be  hastened  by  heat,  as  this  may 
give  rise  to  the  formation  of  substances  that  are  insoluble  in 
alcohol.  The  preparation  should  not  be  touched,  until  it  is 
dry,  with  any  other  instruments  than  such  as  are  made  of 
glass,  porcelain,  or  platinum. 

236.  Characters  of  the  Alumina-Haematein  Stains  (or  of  the  So- 
called  Alum-Haematoxylin  Stains), — The  alum-haematein  lakes 
stain  in  different  tones  of  blue  or  of  red,  according  to  the  com- 
position of  the  staining  solution.  Neutral  or  alkaline  solutions 
give  a  blue  stain ;  acid  solutions  give  a  red  one.  In  order  to 
get  a  l)lue  stain  in  preparations  that  have  come  out  red 
through  the  acidity  of  the  staining  bath,  it  is  a  common 
practice  to  treat  them  with  weak  ammonia,  in  the  belief  that 
the  blue  colour  is  restored  by  neutralisation  of  the  acid  that 
is  the  cause  of  the  redness.  According  to  MAYER,  the 
ammonia  acts  not  by  neutralising  the  acid,  but  by  precipi- 
tating the  alumina,  which  carries  down  the  hgematein  with 
it  (if  no  alumina  were  present  the  colour  would  be  purple, 
not  blue).*  TJie  same  result  can  generally  be  obtained  by 
merely  washing  out  with  common  tap -water,  which  is  usually 
sufficiently  alkaline  (SQUIRE  has  obtained  the  reaction  with 

*  FISCHER,  in  his  Fixirung,  Farbung  u.  Bau  des  Protoplasmus,  pp.  156, 
157,  does  not  admit  this  explanation.  He  proposes  another  one  of  a  highly 
speculative  nature. 


H^MATEIN  (H.EMATOXYLIN)  STAINS.  181 

distilled  water,  free  from  even  a  trace  of  ammonia),  and  can 
be  obtained  with  certainty  by  treatment  with  bicarbonate  of 
-<><hi  or  acetate  of  soda  or  potash.  And  this  is  the  preferable 
cnurse,  as  ammonia  is  certainly  a  dangerous  thing  to  treat 
delicate  tissues  with.  Of  course  this  is  a  different  question 
from  that  of  neutralising  with  an  alkali  tissues  that  have 
been  treated  with  an  acid  to  correct  over-staining.  Here  the 
neutralisation  may  be  indicated  in  the  interest  of  the  pre- 
servation of  the  stain. 

SQUIRE  (Methods,  p.  22)  finds  that  sections  can  be  blued  in 
a  few  seconds  by  treatment  with  a  1  :  1000  solution  of  bicar- 
bonate of  soda  in  distilled  water.  MAYER  holds  that  acetate 
of  potash  is  the  most  inoffensive  reagent  to  take ;  a  strength 
of  O'o  to  1  per  cent,  may  be  taken. 

Several  of  these  solutions  have  a  great  tendency  to  over- 
stain.  Over-stains  may  be  corrected  by  washing  out  with 
weak  acids  (e.  g.  O'l  to  0*2  or  even  0'5  per  cent,  of  hydro- 
chloric acid,  or  with  oxalic  or  tartaric  acid),  but  this  is  not 
favourable  to  the  permanence  of  the  stain.  CAENOY  (La 
Cellule,  xii,  2,  1897,  p.  215)  recommends  iodised  water.  If 
acids  be  used,  it  is  well  to  neutralise  afterwards  with 
ammonia  or  bicarbonate  of  soda  (0*1  per  cent.). 

Bicarbonate  of  soda  may  be  used  for  neutralisation  with 
70  per  cent,  alcohol  as  the  vehicle  (VON  WISTINGBAUSEN,  Mitth. 
Zool.  Stat.  Nea(>el,  x,  1891,  p.  41;  Zeit.  f.  iciss.  Mik.,  x,  4, 
1893,  p.  480). 

Over-staining  may  be  avoided  by  staining  very  carefully 
and  slowly  in  extremely  dilute  solutions.  It  should  be  noted 
that  the  purest  chromatin  stains  are  obtained  by  staining  for 
a  short  time  (sublimate  sections  half  an  hour,  say)  in  solu- 
tions of  medium  strength,  such  as  haemalum  diluted  ten  to 
twenty-fold  with  water.  The  stain  obtained  either  with  very 
strong  solutions,  or  with  the  slow  stain  of  the  dilute  solutions, 
is  at  the  same  time  a  plasma-stain,  which  of  course  may  or 
may  not  be  desired  (MAYER,  in  the  Grundziige,  p.  151,  says 
that  very  dilute  solutions  will  give  a  pure  nuclear  stain  if 
they  have  been  diluted  with  alum- solution,  or  have  been 
'iridified).  Chromosmium  material  will  not  yield  a  pure 
chromatin  stain  unless  it  is  very  fresh  ;  it  is  consequently 
next  to  impossible  to  obtain  the  reaction  with  paraffin  sections 
of  such  material ;  they  constantly  give  a  plasma-stain  in  addi- 


182  CHAPTER   XIII. 

tion  to    the    chromatin    stain,    which    is    not    the    case    with 
sublimate  material. 

The  stain  is  fairly  permanent  in  balsam,  but  is  very  liable 
to  fade  a  little,  and  may  fade  a  great  deal.  If  acids  have 
been  used  after  staining,  great  care  should  be  taken  to  wash 
them  out  thoroughly  before  mounting.  In  aqueous  media  the 
stain  cannot  be  relied  on  to  keep  (this  refers  to  the  old  solu- 
tions :  MAYER  finds  that  his  haematein  preparations  have  kept 
well  for  at  least  some  months  in  glycerin,  if  not  acid,  and, 
with  certain  precautions,  in  balsam).  Turpentine-balsam 
should  not  be  used  (Mayer,  in  litt.}. 

237.  General  Remarks. — We  have  the  coal-tar  colours  for 
staining   sections,  and   we   have    carmine   and   cochineal   for 
staining  in  bulk.      What,  then,  do  we   want  haematein  for  ? 
The  answer  is  that  we  sometimes  want  it  for  staining,  either 
sections  or  in  the  mass,  on  account  of  the  faculty  it  has  of 
staining  tissues   that   have   been  treated   with   chromic   and 
osmic  mixtures.      This   it   does   in  general   better   than  any 
carmine  or  cochineal,  and  sometimes  better  than  any  of  the 
coal-tar  colours.      It  is  also  a  more  powerful  stain  than  car- 
mine ;    and    according  to  the  mode   of    employment   affords 
either  a    chromatin   stain   or   a   sometimes   valuable    plasma- 
stain.      The  chief    haematein    or    haematoxylin  plasma-stains 
are  found  not  amongst  the  alum-haematein  lakes,  but  amongst 
the  other  compounds,  iron  or  chrome  compounds,  etc. 

A.  Alumina-hsematein  Lakes. 

238.  MAYER'S  Haemalum  (MAYEK,  Mittli.  Zool.  Stat.  Neapel, 
x,    1,    1891,    p.    172). — One    grm.   of   the   colouring   matter 
(either  haematein  or  the  ammonia  salt,  §§  234,  235)  dissolved 
with  heat  in  50  c.c.  of  90  per  cent,  alcohol,  and  added  to  a 
solution  of  50  gr.  of  alum  in  a  litre  of  distilled  water.      Allow 
the  mixture  to   cool  and  settle,  and  filter  if  necessary.      Or 
more  recently  (Grundziige,  p.  152),  instead  of  dissolving  the 
haematein  or  salt  in  alcohol,  Mayer    rubs    it   up  in  a  mortar 
with  a  very  little  glycerin. 

It  is  not  necessary  to  conform  exactly  to  the  proportions  given,  and  a 
rough  and  ready  hsemalum  solution  may  be  at  any  time  extemporised  by 
adding  a  few  drops  of  alcoholic  solution  of  hjematein  to  an  alum  solution  of 
any  desired  strength. 


H^EMATEIN    (H^IMATOXYLIN)    STAINS.  183 

A  dark  liquid  of  about  the  tint,  at  first,  of  borax-carmine, 
becoming  more  blue- violet  with  time.  It  stains  equally 
well,  either  at  first,  for  it  is  ripe  from  the  beginning,  or  later. 
Concentrated,  it  stains  sometimes  almost  instantaneously,  or 
in  any  case  very  rapidly.  Diluted  twenty-fold  with  distilled 
water  it  will  still  stain  through  the  tentacles  of  a  Tubularia 
in  an  hour.  (Spring  water  or  tap-water  containing  lime 
must  not  be  used  for  diluting  ;  perhaps  weak  solution  of  alum 
in  distilled  water  is  the  best  means  of  all.)  After  staining, 
sections  may  be  washed  out  either  with  distilled  or  common 
water.  The  solution  is  admira b le  for  staining  in  bulk.  Large 
objects  will,  however,  require  twenty-four  hours'  staining,  and 
should  be  washed  out  for  the  same  time  (this  should  be  done 
with  1  per  cent,  alum  solution  if  a  sharp  nuclear  stain  be 
desired).  All  alum  must  be  carefully  washed  out  of  the 
tissues  before  mounting  in  balsam  ;  and  it  is  well  to  blue  the 
stain  with  tap- water  or  otherwise,  §  236.  The  solution  un- 
fortunately does  not  keep  perfectly,  but  precipitates  and 
becomes  weak  with  age.  When  this  has  occurred,  it  is  well 
to  withdraw  the  quantity  required  for  staining  from  the 
middle  of  the  stock  solution  by  means  of  a  pipette,  which 
should  be  wiped  outside  before  allowing  the  liquid  to  run  out 
of  it.  The  stain  is  generally  a  nuclear  one  ;  in  any  case 
such  may  be  obtained  by  washing  out  with  alum -solution. 
Mayer's  preparations  have  kept  well  in  glycerin  (care  being 
taken  not  to  have  it  acid),  also  in  balsam.  It  is  to  be  noted 
that  if  oil  of  bergamot  be  used  for  clearing,  it  must  be 
thoroughly  removed  by  means  of  oil  of  turpentine  before 
mounting  ;  and  that  oil  of  cloves  is  dangerous.  It  is  best 
(Mayer,  ///  lift.)  to  use  only  xylol,  benzol,  or  chloroform, 
and  to  mount  in  xylol-balsam  or  chloroform-balsam  or  benzol- 
balsam. 

Haemalum  may  be  mixed  with  alum-carmine,  Saurefuchsin, 
or  the  like,  to  make  a  double  staining  mixture ;  but  it  seems 
preferable  to  use  the  solutions  in  succession. 

239.  MAYER'S  Acid  Haemalum  (ibid.,  p.  147,  note). — This 
is  haemalum  with  2  per  cent,  glacial  acetic  acid  (or  4  per 
cent,  common  acetic  acid).  To  be  used  as  the  last,  washing 
out  with  ordinary  water  in  order  to  obtain  a  blue-violet  tint 


184  CHAPTEE   XIII. 

of  stain.      It  is  a  perhaps  even  more  precise    nuclear   stain,  j 
•and  the  solution  keeps  better. 

240.  MAYEE'S  Glychsemalum  (Mitth.  Zool.  Stat.  Neapel,  xii, 
2,  1896,  p.  310). — Haematein  (or  hasmateate  of  ammonia) 
0'4  grm.  (to  be  rubbed  up  in  a  few  drops  of  glycerin  in  a 
mortar  till  it  dissolves)  ;  alum,  5  grms.  ;  glycerin,  30  ;  dis- 
tilled water,  70.  The  stain  is  not  purely  nuclear,  but  may 
be  made  so  by  washing  out  with  alum  solution  or  a  weak 
acid  (§§  236,  238).  The  solution  keeps  admirably. 

241.  HANSEN'S  Solution  (Zool.  Anz.,  1895,  p.  158).— See  last  edition. 
Hansen  oxidises  a  mixture  of  alum   and  hsematoxylin  by  means  of  per- 
manganate of  potash.     He  fancies  that  by  destroying  all  germs  that  may  be 
present  in  the  ingredients,  the  permanganate  should  make  the  solution  keep 
better  than  the  usual   solutions.     As  a  matter  of  fact  it  does  not;  mine 
formed  a  pellicle  and  strong  precipitate  in  a  few  days,  and  the  same  was 
found  at  the  Naples  station  (MAYER,  in  Hit.}.     See  further  the  remarks  of 
MAYER  on  this  process  in  Mitth.  Zool.  Stat.  Neapel,  xii,  1896,  p.  309,  or  the 
Grundziige,  p.  153. 

242.  HARRIS'S  Solution  (Micr.  Bull.,  xv,  1898,  p.  47  ;  Journ.  Roy.  Mic. 
Soc.,  1899,  p.  236). — Aluin-hsernatoxylin  solution  ripened  by  addition  of 
mercuric  oxide. 

243.  Bohmer's  Haematoxylin  (Arch.f.  mik.  Anat.,  iv,  1868,  p.  345  ; 
Aerzt.   IntelligenzbL,    Baiern,    1865,   p.    382). — Make    (A)    a   solution   of 
hsematox.  cryst.  1  part,  alcohol  (absolute)  12  parts  and  (B)  alum  1  part, 
water  240.     For  staining,  add  two  or  three  drops  of  A  to  a  watch-glassful 
of  B. 

The  alcoholic  solution  of  hsematoxylin  ought  to  be  old  and  brown  (§  232)- 
I  consider  this  stain  to  "be  of  merely  historical  interest. 

244.  Delafield's  Heematoxylin  (Zeit.  f.  wiss.  Mik.,  ii,  1885,  p.  288: 
frequently  attributed  erroneously  to  GRENACHEROI-  PRUDDEN). — To  400  c.c. 
of  saturated  solution  of  ammonia-alum*  add  4  grms.  of  hsematox.  cryst. 
dissolved  in  25  c.c.  of  strong  alcohol.     Leave  it  exposed  to  the  light  and  air 
in  an  unstoppered  bottle  for  three  or  four  days.     Filter,  and  add  100  c.c.  of 
glycerin  and  100  c.c.  of  methylic  alcohol  (CH40).     Allow  the  solution  to 
stand  until  the  colour  is  sufficiently  dark,  then  filter  and  keep  in  a  tightly 
stoppered  bottle. 

This  solution  keeps  well,— it  maybe  said  to  keep  for  years.     It  is  well  to 
allow  it  to  ripen  for  at  least  two  months  before  using  it. 

For  staining,  enough  of  the  solution  should  be  added  to  pure  water  to 

*  Ammonia-alum  dissolves  in  about  11  parts  0  water. 


H^IMATEIN  (H^IMATOXYLIN)  STAINS.  185 

make  a  very  dilute  stain  ;  and  even  then  care  should  be  taken  not  to  leave 
objects  too  long  in  the  fluid.     It  is  an  extremely  powerful  stain. 

BITSCHLI  (Unters.  ilb.  mikroscopische  Schdume  u.  das  Protoplasma, 
etc.,  1892 ;  Zeit.  f.  wiss.  Mik.,  ix,  2,  1892,  p.  197)  recommends,  under  the 
name  of  "  acid  haematoxylin,"  solution  of  Delafield  very  strongly  diluted, 
and  with  enough  acetic  acid  added  to  it  to  give  it  a  decidedly  red  tint. 
This  gives  a  sharper  and  more  differentiated  nuclear  stain  than  the  usual 
solution. 


245.  Ehrlich's  Acid  Haematoxylin  (Zeit.  f.  wiss.  Mile.,  1886, 
p.  150).— 

Water 100  c.c. 

Absolute  alcohol         .....     100   ,, 

Glycerin 100   „ 

Glacial  acetic  acid      .         .         .         .  10   „ 

Haematoxylin 2  grms. 

Alum  in  excess. 

Let  the  mixture  ripen  in  the  light  (with  occasional  admission  of  air) 
until  it  acquires  a  dark  red  colour.  It  will  then  keep,  with  constant 
staining  power,  for  years,  if  kept  in  a  well- stoppered  bottle.  Sections  are 
stained  in  a  few  minutes.  It  is  stated  that  the  solution  ia  also  very 
appropriate  for  staining  in  bulk,  as  over-staining  does  not  occur. 

Of  all  the  old  formulae  1  have  tried,  this  is  the  one  that  has  given  me 
the  sharpest  chromatin  stain. 

MANN  (Zeit.  f.  wiss.  Mik.,  xi,  4,  1895,  p.  487)  makes  ap  this  stain  with 
an  equal  quantity  of  haeinatein  instead  of  haematoxylin. 

MATES  (Grundzuge,  p.  154)  finds  that  this  is  too  much,  and  makes  the 
mixture  overstain  ;  0*4  grm.  of  hsematein  is  quite  enough. 

246.  MAYER'S  Haemacalcium  (Mitth.  Zool.  Stat.  Neapel,  x, 
1,  1891,  p.  182). — Haemateiii  (or  haemateate  of  ammonia,  §§ 
'234,  235),  1  grm.  ;  chloride  of  aluminium,  1  grm.  ;  chloride 
of  calcium,  50  grms. ;  glacial  acetic  acid,  10  c.c.  (or  common 
acetic  acid,  20  c.c.)  ;  70  per  cent,  alcohol,  600  c.c..  Rub  up 
finely  together  the  first  two  ingredients,  add  the  acid  and 
alcohol,  dissolve  either  cold  or  with  heat ;  lastly  add  the 
chloride  of  calcium. 

A  reddish -violet  liquid.  If  the  objects  stain  in  too  red  a 
tone  they  may  be  treated  with  a  aulution  (of  about  2  per 
cent.)  of  chloride  of  aluminium  in  70  per  cent,  alcohol,  or 
with  a  0'5  to  1  per  cent,  solution  of  acetate  of  soda  or  potash 
in  absolute  alcohol  ;  but  washing  with  neutral  alcohol  will 
generally  suffice. 

The  solution  is  not  perfectly  stable,  but  in  course  of  time 
(Mitth.  a.  d.  Zool.  Stat.  Ntapel,  x,  3,  1892,  p.  499)  turns  blue 


186  CHAPTER  XIII. 

and  precipitates.  To  avoid  this  the  mixture  should  be  made 
up  in  two  separate  bottles,  each  containing  half  of  the  alcohol 
and  of  the  acid,  and  one  containing  besides  all  the  calcium 
chloride,  the  other  all  the  haematein  and  all  the  aluminium 
chloride,  equal  quantities  being  taken  from  each  when  re- 
quired for  staining. 

With  certain  objects  this  solution  does  not  penetrate  well, 
the  stain  being  confined  to  their  superficial  parts.  This  may 
be  remedied  by  acidifying  the  solution,  or,  which  is  better, 
by  leaving  the  objects  for  some  time  before  staining  in  acid 
alcohol.  Anyway  objects  ought  NOT  to  have  an  alkaline  re- 
action. If  these  precautions  be  taken,  it  will  not  be  necessary 
to  use  acid  for  washing  out.  For  some  objects  also  (e.  g. 
Hydroida)  the  penetrating  effect  is  enhanced  by  diluting  the 
solution  with  one  third  volume  of  glycerin,  or  by  increasing 
the  proportion  of  aluminium  chloride  up  to  about  eight  times 
that  of  the  haematein. 

The  solution  is  not  recommended  as  giving  as  good  results 
as  hasmalum, — as  a  stain  it  is  distinctly  inferior  ;  and  Mayer 
is  of  opinion  that  no  alcoholic  haematein  solution  can  be  made 
to  give  as  precise  a  stain  as  the  aqueous  solutions.  He 
recommends  it  merely  as  a  substitute  for  Kleinenberg's  (in 
cases  in  which  an  alcoholic  haematein  stain  seems  indicated), 
as  being  convenient,  easy  to  prepare,  and  constant  in  its 
effects,  none  of  which  qualities  belong  to  Klemenberg's 
formula. 

247.  APATHY'S  Haematein  Mixture  I  A  (Mitth.  Zool.  Stat. 
Neapelj  xii,  1897,  p.  712). — Make  (A)  a  solution  of  9  per 
cent,  alum,  3  per  cent,  glacial  acetic  acid,  and  0*1  per  cent, 
salicylic  acid  in  water,  and  (B)  a  1  per  cent,  solution  of 
haematoxylin  in  70  per  cent,  alcohol,  preserved  for  six  to 
eight  weeks  in  a  bottle  not  quite  full.  Mix  one  part  of  A 
with  one  of  B  and  one  of  glycerin.  The  solution  will  keep 
for  years,  and  stains  either  sections  or  material  in  bulk. 
Apathy  uses  it  for  staining  nerve  "  primitive-fibrils  ;"  it  isr 
therefore,  not  a  purely  nuclear  stain. 

248.  KLEINENBERG'S  Haematoxylin  (Quart.  Journ.  Micr.  Sci.,  Ixxiv, 
1879,  p.  208). — Highly  irrational  and  very  inconstant  in  its  composition  and 
its  effects;  see  earlier  editions;  also  the  elaborate  criticism  of  MAYER 


HAMATEIN    (HJ1MATOXYLIN)    STAINS.  187 

(Mitth.  a.  (I  Zool.  Stat.  zu  Neapel  x,  1, 1891,  p.  174),  and  that  of  SQUIRE, 
in  his  Methods  and  Formulae,  p.  25,  and  the  alternative  formulae  of  SQUIRE 
(loc.  cit.)  and  of  VON  WISTINGHAUSEN  (Mitth.  Zool.  Stat.  Neapel,  x,  1891, 
p.  41  ;  Zeit.f.  wiss.  Mik.,  x,  4,  1893,  p.  479). 

249.  BURCHARDT'S  Pyroligneous  Acid  Hsematoxylin  (Arcli.f.  mik. 
Anut.,  liii,  1898,  p.  232;  Zeit.f.  wiss.  Mik.,  xv,  4,  1899,  p.  453;  Journ. 
Roy.  Mic.  Soc.,  1899,  p.  453)  would  seem  to  be  superfluous  at  least. 

250.  Other  Alumina-haematein    Solutions. — A    large    number   of 
suppressed  receipts  will  be  found  given  in  the  earlier  editions. 


B.  Other  Hsematein  or  Hsematoxylin  Compounds. 

251.  Introduction. — There  remain  to  be  described  the  stains 
derived  from  the  compounds-  of  haematein  or  haematoxylin 
with  iron,  copper,  chrome,  vanadium,  and  molybdenum. 
Only  those,  however,  of  general  applicability  will  be  de- 
scribed in  this  place  ;  those  which  are  only  employed  as 
specific  stains  for  nervous  tissue  (Weigert-Pal,  etc.)  finding 
their  place  under  Neurological  Methods.  The  iron  stains  can 
only  be  employed  for  sections  ;  the  chrome  stains  may  also 
be  used  on  material  in  bulk. 

252.  R.  HEIDEXHAIN'S  Chrome  Heematoxylin  (Arch.  f.  mik.  Anat.y 
xxiv,  1884,  p.  468,  and  xxvii,  1886,  p.  383). — Stain  for  twelve  to  twenty- 
four  hours  in  a  ^  per  cent,  solution  of  hsematoxylin  in  distilled  water. 
Soak  the  objects  for  the  same  length  of  time  in  a  0'5  per  cent,  solution  of 
neutral  chromate  of  potash,  which  should  be  changed,  if  necessary,  several 
times.  Wash  out  the  excess  of  chromate  with  water. 

The  above  is  a  slightly  modified  form  of  the  original  process,  in  which 
staining  was  done  in  a  stronger  haematoxylin  solution  (0'5  to  1  per  cent.), 
and  bichromate  was  used  for  washing  out  instead  of  neutral  chromate.  The 
more  recent  process  gives  a  sharper  chromatin  stain. 

The  stain  succeeds  best  with  alcohol  or  picric  acid  objects,  but  it  will 
succeed  with  chromic  objects  if  they  have  been  very  well  washed,  or  with 
material  fixed  in  Flemming's  mixture. 

Objects  that  have  been  fixed  in  corrosive  sublimate  ought  to  be  very  care- 
fully washed  out  with  iodine,  or  the  like  (see  §  66),  as  neutral  hsematoxylin 
forms  a  black  precipitate  with  any  excess  of  sublimate  that  may  remain  in 
the  tissues  (see  TORNIER,  in  Arch.f.  mik.  Anat.,  1886,  p.  181). 

The  stain  is  black  to  grey.  It  is  a  sharp  stain,  remarkably  rich  in  detail. 
It  is  a  plasma-stain  as  well  as  a  chromatin  stain. 

The  process  is  adapted  to  staining  in  bulk.  You  can  decolour  the  objects 
to  any  extent  by  prolonging  the  washing  in  the  chromate. 


188  CHAPTER    XIII. 

The  method  may  be  varied  by  washing  out  after  staining  with  alum  solu- 
tion (1  per  cent.)  instead  of  a  chromate.  In  this  case  the  stain  will  be  blue. 

253.  APATHY'S  Modification  of  Heidenhain's  Process  (Zeit.f.  wiss. 
Mik.,  v,  1, 1888,  p.  47).— This  is  an  alcoholic  method. ,  Stain  in  a  1  percent, 
solution  of  hcematoxylin  in  70  or  80  per  cent,  alcohol.  Wash  out  (for 
•"  thin  "  sections,  i.  e.  sections  of  10  to  15  /i,  half  the  time  of  staining — for 
"  thicker"  sections  of  25  to 40  /*  twice  the  time  of  staining)  in  1  per  cent, 
solution  of  bichromate  of  potash  in  70  to  80  per  cent,  alcohol. 

The  bichromate  solution  is  conveniently  prepared  by  mixing  one  part  of  a 
5  per  cent,  aqueous  solution  with  about  four  parts  of  80  to  90  per  cent, 
alcohol.  The  mixture  should  be  made  immediately  before  using,  and  should 
be  kept  from  the  light  (light  precipitates  it)  during  the  process  of  decolora- 
tion, and  should  also  be  changed  for  fresh  several  times  during  the  process. 
After  the  differentiation  of  the  colour  has  been  accomplished,  the  objects 
should  be  thoroughly  washed  (still  in  the  dark)  in  several  changes  of  70  per 
cent,  alcohol. 

Preparations  made  in  this  manner  are  said  to  be  more  transparent  and 
better  preserved  than  those  made  by  Heidenhain's  process. 

For  celloidin  series  of  sections,  Apathy  (Zeit.  /.  wiss.  Mik.,  vi,  2,  1889, 
p.  170)  stains  in  the  haematoxylin  solution  as  above  for  ten  minutes  ;  then 
removes  the  excess  of  hsematoxylin  fluid  from  the  sections  by  means  of 
blotting-paper,  and  brings  the  series  for  five  to  ten  minutes  into  70  per  cent, 
.alcohol  containing  only  a  few  drops  of  a  strong  (5  per  cent. ^solution  of 
bichromate.  This  must  be  done  in  the  dark.  If  the  hjEinatoxylin  be  not 
removed  with  blotting-paper  as  described,  the  celloidin  will  take  the  stain. 
The  sections  should  appear  steel-blue  to  steel-grey. 

254.  BENDA'S  later  Iron  Haematoxylin  (Verh.  <L  Aimt.  Ges., 
vii,  1,  1893,  p.  161  ;  Zeit.  f.  wits.  Mik.,  xi,  1,  18^4,  p.  69; 
for  his  earlier  method  see  Arch.  Anat.  PJiys.,  Pliys.  Abth., 
1886,  p.  564). — Tissues  fixed  in  any  way  may  be  employed. 
Sections  are  mordanted  for  twenty-four  hours  in  Liquor ferri 
sulphurici  oxidati,  P.  Gr._,  diluted  with  one  or  two  volumes  of 
water  (this  preparation  consists  of  sulphate  of  iron  80  parts, 
water  40,  sulphuric  acid  15,  and  nitric  acid  18,  and  contains 
10  per  cent,  of  Fe).  They  are  then  well  washed,  first  with 
.distilled  water,  then  with  tap-water,  and  are  brought  into  a 
i  per  cent,  solution  of  haematoxylin  in  water,  in  which  they 
remain  till  they  have  become  thoroughly  black.  They  are 
then  washed  and  differentiated.  The  differentiation  may  be 
done  either  in  30  per  cent,  acetic  acid,  in  which  case  the  pro- 
gress of  the  decoloration  must  be  watched ;  or  in  a  weaker 
acid,  which  will  not  require  watching  ;  or  in  the  sulphate 
solution  strongly  diluted  with  water. 


HJEMATEIN  (H^EMATOXYLIN)  STAINS.  189- 

rriiis  process  is  applicable  to  all  sorts  of  organs,  and  gives 
in  particular  excellent  images  of  axis-cylinders,  and  of  the 
achromatic  figure  of  cell-division.  It  is  sometimes  useful  to 
add  a  second  stain  with  Saurefuchsin,  or  Bordeaux.  If  the 
iron  solution  be  taken  for  the  differentiation,  it  should  be 
taken  extremely  diluted  (of  a  very  pale  straw-colour),  and 
the  progress  of  the  differentiation  Avatched ;  as  if  it  be  only 
diluted  about  tenfold,  for  instance,  the  decoloration  is  ex- 
tremely rapid. 

The  stain  is  nuclear  or  nuclear  and  plasrnatic  according  to 
the  amount  of  differentiation,  and  is  one  of  the  most  splendid 
stains  known.  The  tone  varies  somewhat  between  dark  blue 
and  dead  black  (see  next  §).  After  most  carefully  com- 
paring it  with  that  of  M.  Heidenhain  (next  §)  I  find  that  in 
result  the  two  are  absolutely  identical,  Benda's  process  having 
the  advantage  that  his  Liq.  fe.  sulph.  keeps  indefinitely, 
which  Heidenhain' s  ferric  solution  does  not.  Either  of  these 
processes,  when  successful,  gives  a  stain  of  an  optical  quality 
that  is  peculiarly  suited  to  the  employment  of  high  micro- 
scopic powers,  and  will  allow  of  the  employment  of  deeper 
eye-piece*  than  any  other  stain  known  to  me.  Both  the  pro- 
cesses are  somewhat  crotchety,  and  I  have  met  with  material 
with  which  it  has  been  impossible  to  obtain  the  correct  stain 
(probably  on  account  of  some  peculiarity  in  the  fixation). 

255.  Iron  Haematoxylin  (M.  HEIDENHAIN,  "  Uber  Kern  und 
Protoplasms,"  in  Festschr.  Herrn.  Geheimr.  A.  v.  Kolliker,  etc., 
geu'idm.,  1892,  p.  118). — Sections  are  treated  from  half  an 
hour  to  at  most  two  or  three  hours  with  a  1'5  to  4  per  cent, 
solution  of  ferric  alum  (ammonio-ferric  sulphate).  By  this 
is  meant  the  double  salt  of  the  sesquioxide  of  iron  (NH4)2 
Fe2(S04)4,  in  clear  violet  crystals;  the  double  salt  of  the- 
protoxide,  or  salt  of  MOHR  in  green  crystals,  will  not  serve. 
If  the  crystals  have  become  yellow  and  opaque,  they  have 
gone  bad,  and  should  be  rejected.  They  ought  to  be  kept 
in  a  stoppered  bottle,  and  the  solution  should  be  made  in .  the 
cold  (Arch.f.  mik.  Anat.,  xliii,  3,  1894,  pp.  431,  435).  The 
sections  are  then  washed  with  water  and  stained  for  half  an 
hour  in  an  aqueous  solution  (of  about  0*5  per  cent.)  of 
hasmatoxylin.  (Haematoxylin  is  stated  by  Heidenhain  k> 
give  better  results  than  haematein.)  They  are  then  rinsed 


190  CHAPTER   XIII. 

with  water,  and  again  treated  with  the  iron  solution,  which 
slowly  washes  out  the  stain.  The'  progress  of  the  differen- 
tiation ought  to  4)e  Controlled  under  the  microscope.  The 
sections  should  t'o'^^s  end  be  removed  from  time  to  time 
from  the  alum  solution,  and  put  into  tap-water  whilst  they 
are  being  examined.  This  is  favourable  to  the  stain.  As 
soon  as  a  satisfactory  differentiation  has  been  obtained,  the 
preparations  are  washed  for  at  least  a  quarter  of  an  hour  in 
running  water,  but  not  more  than  an  hour,  and  mounted. 
The  results  differ,  in  Heidenhain's  view,  according  to  the 
duration  of  the  treatment  with  the  iron  and  the  stain.  If 
the  baths  have  been  of  short  duration,  viz.  not  more  than 
half  an  hour  in  the  iron  and  as  much  in  the  stain,  blue 
preparations  will  be  obtained.  These  show  a  very  intense 
and  highly  differentiated  stain  of  all  nuclear  structures, 
cytoplasmic  structures  being  pale.  If  t'he  baths  in  the  iron 
.and  in  the  stain  have  been  prolonged  (twelve  to  eighteen 
"hours),  and  the  subsequent  differentiation  in  the  second  iron 
bath  also  duly  prolonged,  Hack  preparations  will  result.  These 
show  chromosomes  stained,  "  central  corpuscles  "  stained  in- 
tensely black,  cytoplasm  sometimes  colourless,  sometimes  grey, 
in  which  case  achromatic  spindle-fibres  and  cell-plates  are 
stained,  connective -tissue  fibres  black,  red  blood- corpuscles 
black,  micro-organisms  sharply  stained,  striated  muscle  very 
finely  shown. 

Dr.  MAYER,  writing  to  me,  doubts  that  the  blue  or  black 
tone  is  conditioned  by  the  duration  of  the  mordanting  and 
staining  baths ;  and  my  observations  confirm  this  view. 

I  most  highly  recommend  this  stain,  which,  like  BENDA'S, 
is  one  of  the  very  finest  I  am  acquainted  with.  It  may  be 
used  either  with  sublimate  or  alcohol  material,  or  after  liquid 
of  Flemming.  The  process  is  extremely  easy  to  manage. 
It  is  only  applicable  to  sections,  which  should  be  thin,  best 
not  more  than  8  ju  in  thickness.  The  preparations  are  per- 
fectly permanent.  It  has  been  said  that  this  process  frequently 
gives  rise  to  amorphous  precipitates  in  the  tissues.  I  find  that 
it  does  sometimes,  but  not  to  any  very  injurious  extent. 

Later  (Zeit.  f.  wiss.  Mile.,  xiii,  1896,  p.  186),  Heidenhain  gives  further 
instructions  for  the  employment  of  this  stain  in  the  study  of  his  "  central 
corpuscles."  All  alcohol  should  be  removed  from  the  tissues  by  means  of 
distilled  water  before  bringing  them  into  the  mordant.  This  should  be  a 


HJEMATEIN  (HJSMATOXYLIN)  STAINS.  191 

2£  per  cent,  solution  of  ferric  alum,  not  weaker.  Leave  the  sections 
therein  (fixed  to  slides  by  the  water  method,  §  182)  for  six  to  twelve  hours, 
or  at  least  not  less  than  three.  Keep  the  slides  upright  in  the  mordant,  not 
lying  flat.  Wash  out  well  with  water  before  staining.  Stain  in  a  "  ripened  " 
hsematoxylin  solution,  i.  e.  one  that  has  stood  for  four  weeks  (of  course  if 
,  you  make  it  up  with  the  ripened  brown  alcoholic  solution  recommended 
§  232,  sub  fin.,  this  will  be  superfluous).  Stain  for  twenty-four  to  thirty- 
six  hours.  Use  the  same  staining  solution  over  and  over  again  until  it 
becomes  spoilt;  for  the  solution  after  having  been  used  gives  a  more  ener- 
getic stain,  owing  to  its  containing  a  trace  of  iron  brought  over  by  the» 
sections.  Differentiate  in  a  2|  per  cent,  solution  of  ferric  alum.  Rinse  for 
ten  minutes  in  running  water,  clear  with  xylol,  not  with  any  essential  oil, 
and  mount  in  xylol-balsam.  See  further  on  this  subject  under  "  Cytological 
Methods." 

256.  Iron    Hsematoxylin   (BuxscHLi,   Unters.   uber  mikroskopische 
Schiiume  u.  das  Protoplasma,  etc.,  1892  ;  Zeit.f.  wiss.  Mik.,  ix,  2,  1892, 
p.  197). — Sections  treated  with  a  weak  brown  aqueous  solution  of  ferric 
acetate,  washed  with  water,  and  stained  in  05  per  cent,  aqueous  solution  of 
haematoxylin.     This  treatment  gives  a  blue-black  or  brown-black  stain  of 
extraordinary  intensity.     The  process  was   used  by  Biitschli  for  staining 
sections,  1  /*  in  thickness,  of  Protozoa.     It  does  not  appear  to  be  of  general 
applicability. 

257.  JANSSENS'  Iron  Haematoxylin  ("  He'matoxyline  noire;"  La 
Cellule,  xiv,  1,  1897,  p.  207). — A  similar  mixture  to  that  of  DELAFIELD, 
§  244,  ferric  alum  being  taken   instead  of  ammonia  alum,  the  rest  as  in 
Delafield's.     A  progressive  stain,  nuclear. 

258.  BENDA'S  Copper  Haematoxylin  (Arch.  f.  mik.  Anat.,  xxx,  1887, 
p.  49).     See  last  edition.     According  to  my  experience,  not  to  be  compared 
with  iron  hsematoxylin,  and  superfluous. 

259.  MALLORY'S    Phospho-molybdic   Acid  Heematoxylin   (Anat. 
Anzeig.,  1891,  p.  375  ;  see  also  Zeit.  f.  wiss.  Mik.,  viii,  3,  1891,  p.  341). — 
One  part  of  10  per  cent,  phospho-molybdic  acid  solution,  1  part  hsematoxylin, 
100  parts  water,  and  6  to  10  pails  chloral  hydrate.     Let  the  solution  ripen 
for  a  week  in  sunlight,  and  filter.     This  stain  is  recommended  for  prepara- 
tions of  central  nervous  system,  but  has  been  found  useful  in  other  cases. 
Sections  should  be  stained  for  from  10  minutes  to  1  hour,  and  washed  out  in 
two  or  three  changes  of  40  to  50  per  cent,  alcohol.     Dehydrate  and  mount 
as  usual.    Celloulin  remains  colourless.    The  stain  is  blue,  and  in  its  general 
effect  something  like  a  nigrosin  stain.    Besides  ganglion-cells  and  glia-cells, 
axis-cylinders  are  stained,  also  many  other  tissue-elements.     It  is  necessary 
that  the  solution  be  saturated  with  hsematoxylin  in  order  to  obtain  the  best 
results ;  if  a  good  stain  be  not  obtained  at  once,  more  hsematoxylin  must  be 
added. 

See  also  RIBBEET  (Centralb.  f.  allg.  Path.,  vii,  1896,  p.  427  ;  Zeit.  f.  iviss? 
Mik.,  xv,  1,  1898,  p.  93). 


192  CHAPTER    XIII. 

SARGENT  (Anat.  Anz.,  xv,  1898,  p.  214)  quotes  this  stain,  preceded  by 
mordanting  for  twenty-four  hours  in  5  per  cent,  sulphate  of  copper,  as 
KENYON'S. 

260.  MALLOEY'S    Phosphotungstic    Acid    Haematoxylin    (Journ. 
Exper.  Med.,  ii,  1897,  No.  5,  p.  531). — Dissolve  0*1  grm.  hsematoxylin  in  a 
little  hot  water,  and  when  cool  add  to  100  c.c.  of  1  per  cent,  solution  of 
phosphotungstic  acid  (MEBCK).     Stain  for  2  to  24  hours.     Nuclei  blue, 
connective-tissue  substances  pink. 

261.  MINOT'S  Haematoxylin  Methods  (Zeit.  f.  wiss.  Mik.,  iii,  2,  1886, 
p.  177). 

See  also  BOLTON,  Journ.  Anat.  and  Phys.,  xxxii,  1898,  p.  247,  and  xxxiii, 
1899,  p.  297. 

» 


CHAPTER  XIV. 

ON    STAINING    WITH   COAL-TAR   COLOURS. 

262.  Basic,  Acid,  and  Neutral  Coal-tar  Colours. — Histologists 
generally  conceive  of  tlie  coal-tar  colours  as  divided  into 
three  groups,  according  to  a  principle  of  classification  founded 
on  chemical  considerations,  and  introduced  into  histological 
literature  by  EHRLICH  (Zeit.  klin.  Med.,  1,  1880,  p.  555; 
Verh.  d.  Berl.  Phijs.  Ges.,  May  16th,  1879;  in  REICHERT  AND 
Du-Bois  RKYMOND'S  Arch.  f.  Anat.  u.  P%*.,  Phys.  Abth., 
1879,  p.  571).  These  three  groups  are  those  of  the  basic 
colours,  the  acid  colours,  and  the  neutral  colours.  By  a 
"  basic  "  colour  is  meant  a  compound  in  which  the  colouring 
principle  or  molecular  group  to  which  the  compound  owes 
its  colouring  properties  exists  as  or  chemically  plays  the 
part  of  a  base  combined  with  a  colourless  acid.  For  instance, 
f  uchsin  or  magenta  is  a  basic  colour.  It  is  the  hydrochloride 
of  rosanilin,  and  its  colouring  properties  are  due  to  the 
rosanilin  which  exists  as  a  base  in  the  compound,  and  not  to 
the  hydrochloric  acid  of  the  compound.  By  an  "  acid " 
colour  is  meant  a  compound  in  which  the  colouring*  principle 
exists  as  or  plays  the  part  of  an  acid.  The  dye  known  as 
acid  f  uchsin  or  acid  magenta  (Sauref  uchsin)  is  an  "acid" 
colour.  It  is  the  soda  salt  of  di-  or  tri-sulphoconjugated 
rosanilin,  that  is  of  rosanilin  di-  or  tri-sulphonic  acid,  and  its 
colouring  properties  are  due  to  the  rosanilin  which  exists  as 
an  acid  in  the  compound,  and  not  to  the  soda.  Or  to  take  a 
simpler  case,  picrate  of  ammonia  is  an  "  acid "  colour  in 
Ehrlich's  sense,  and  its  colouring  properties  are  evidently 
due  to  the  picric  acid  in  it,  and  not  to  the  ammonia.  The 
neutral  colouring  matters  form  a  very  small  group  ;  the  only 
example  that  I  can  find  mentioned  in  BENEDIKT  and  KNECHT'S 
Chemistry  of  the  Coal-tar  Colours  being  artificial  indigo, 

13 


194  CHAPTER    XIV. 

obtained  from  propiolic  acid.  It  appears,  however,,  to  be 
established  that  neutral  colours  are  frequently  formed  by  the 
mixture  of  the  solutions  of  an  acid  colour  and  a  basic  colour. 
They  are  generally  insoluble  in  pure  water,  and  hence  pre- 
cipitate when  the  mixture  is  made,  but  may  be  got  to  re- 
dissolve  by  adding  an  excess  of  the  acid  colour,  or  of  the 
basic,  and  are  always  soluble  in  alcohol. 

See  further  as  to  the  "  neutral"  colours  thus  obtained,  BOSIN,  "  Ueber 
eine  neue  Gruppe  der  Anilinfarbstoffen,"  in  Berliner  "klin.  Wochenschr., 
xii,  1898,  p.  251 ;  Zeit.  f.  wiss.Mik.,  xvi,  2, 1899,  p.  223  ;  Journ.  Roy.  Mic. 
Soc.,  1899,  p.  547. 

Now,  according  to  Ehrlich,  the  basic  colours  are  in  general 
chromatin  stains, — that  is,  the^r  have  a  special  affinity  for 
the  element  of  nuclei  known  as  chromatin,  so  that  they  are 
mostly  sharp  nuclear  stains.  The  acid  colours,  on  the  other 
hand,  are,  according  to  him,  in  general  plasma  stains, — that 
is,  they  have  a  special  affinity  for  cytoplasm  and  intercellular 
substances.  And  lastly  the  neutral  colours  exhibit  special 
affinities  for  certain  cell-contents  ;  amongst  them  are  found 
some  important  granule  stains. 

I  think  that  that  is  a  generalisation  which  requires  to  be 
supplemented  by  a  good  deal  of  explanation  and  restriction. 
In  practical  histology  we  have  to  take  account  not  only  of 
the  "  affinities  "  of  a  dye  for  this  or  that  cellular  element,  as 
they  are  manifested  in  progressive  staining  under  narrowly 
limited  conditions ;  we  have  also  to  take  account  of  the 
resistance  of  the  stain  to  the  liquids  employed  for  washing, 
for  dehydration,  for  clearing  ;  in  short,  we  have  to  take  into 
account  the  way  in  which  the  dye  behaves  when  employed 
as  a  regressive  stain.  This  is  of  peculiar  importance  in  the 
case  of  the  coal-tar  colours,  seeing  that  they  are  largely 
used  for  the  regressive  staining  of  sections  destined  to  be 
dehydrated  by  alcohol  and  mounted  in  balsam.  Now  Erhlich's 
experiments  take  no  account  of  these  conditions.  He  worked 
with  "  cover-glass  preparations "  of  isolated  cells,  such  as 
blood  and  lymph  cells,  and  was  thus  able  to  avoid  the  pro- 
longed washing  necessary  for  most  sections,  and  to  suppress 
altogether  the  dehydration  by  alcohol,  his  cover-glass  prepara- 
tions being  simply  dried  after  staining  in  a  stove.  In  con- 
sequence, his  chemical  categories  of  basic  colours  and  acid. 


ON   STAINING    WFTH    COAL-TAIi    COLOURS.  195 

il  to  correspond  precisely  to  the  technical  categories 
of  chromatin  stains  and  plasma  stains. 

For  instance,  orange  is  an  acid  colour;  but  used  as  a  re- 
gressive stain  I  find  it  will  give  a  very  sharp  stain  of  chro- 
matiii :  it  cannot,  therefore,  be  classed  as  a  mere  plasma 
<taiii,  though  it  is  also  a  very  good  plasma  stain.  Saure- 
fuchsin  is  a  very  acid  colour.  It  behaves  in  general  as  a 
decided  plasma  stain.  But  used  as  a  regressive  stain  it 
sometimes,  under  conditions  which  I  am  not  able  to  specify, 
gives  a  very  vigorous  stain  of  chromatin.  Safranin  is  a 
basic  colour,  but  by  the  use  of  appropriate  mordants  it  can 
be  made  to  behave  as  a  plasma  stain.  Methylen  blue  is  a 
basic  colour.  But,  as  is  well  known,  when  employed  according 
to  the  method  worked  out  by  Ehrlich  for  the  so-called  intra- 
uitam  staining  of  nerves,  it  affords  a  stain  that  is  essentially 
plasmatic,  such  staining  of  nuclei  as  may  occur  in  this  pro- 
cess being  an  accidental  epiphenomenon.  Nigrosin  is,  ac- 
cording to  Ehrlich,  an  acid  colour,  and  should  therefore  be 
essentially  a  plasma  stain.  Yet  I  find  that,  used  as  a 
regressive  stain  in  the  same  way  as  safranin,  it  gives  a 
vigorous  chromatin  stain,  cytoplasm  being  only  faintly 
coloured.  Bordeaux  is  an  acid  colour,  but  it  stains  chromatin 
as  Avell  as  cytoplasm  :  and  many  similar  cases  might  be  men- 
tioned. Indeed,  it  is  not  too  much  to  assert  that  there  is 
hardly  any  colour,  either  basic  or  acid,  that  may  not  be 
made  to  afford  either  a  chromatin  stain  or  a  plasma  stain, 
according  to  the  way  in  which  it  is  employed. 

It  would  seem,  therefore,  that  Ehrlich' a  generalisation 
does  not  hold  good  as  a  statement  of  the  behaviour  of  tar 
-colours  when  employed  for  staining  sections  in  the  usual  way. 
It  is  roughly  true  that  the  basic  colours  are  in  general 
chromatin  stains,  and  the  acid  colours  in  general  plasma 
stains ;  but  the  rule  is  subject  to  many  exceptions. 

263.  Progressive  and  Regressive  Coal-tar  Stains. — Very  few 
tar  colours  give  a  precise  nuclear  or  chromatin  stain  by  the 
progressive  or  direct  method  (§  199).  Two  of  them — methyl 
green  and  Bismarck  brown — are  pre-eminently  chromatin 
stains.  Many  of  the  others — for  instance,  safranin,  gentian, 
and  especially  dahlia — may  be  made  to  give  a  nuclear  stain 
with  fresh  tissues  by  combining  them  with  acetic  acid ;  but 


196  CHAPTER   XIV. 

in  ninety-nine  cases  out  of  a  hundred  are  not  so  suitable  for 
this  kind  of  work  as  the  two  colours  first-named,  which 
practically  form  a  class  apart. 

Again,  very  few  tar  colours  give  a  pure  plasmatic  stain 
(one  leaving  nuclei  unaffected) .  The  majority  give  a  diffuse 
stain,  which  in  some  few  cases  becomes,  by  the  application 
of  the  regressive  or  indirect  method  (§  199),  a  most  precise 
and  splendid  chromatin  stain. 

The  regressive  staining  method  will  form  the  subject  of 
the  present  chapter,  and  the  chromatin  stains  will  be  treated 
of  in  the  next  chapter,  the  plasma  stains  being  reserved  for 
treatment  in  a  later  chapter. 


General  Directions  for  the  Regressive   Staining  Method, 
as  applied  to  Coal-tar  Colours* 

264.  Staining1. — Sections  only,  or  material  that  is  thin 
enough  to  behave  like  sections,  such  as  some  membranes,  can 
be  stained  by  this  method. 

The  solutions  employed  are  made  with  alcohol,  water,  or 
anilin,  or  sometimes  other  menstrua,  according  to  the  solubility 
of  the  colour.  There  seems  to  be  no  special  object  in  making 
them  with  alcohol  if  water  will  suffice,  the  great  object  being 
to  get  as  strong  a  solution  as  possible.  Indeed,  the  solutions 
made  with  strong  alcohol  are  found  not  to  give  quite  such 
good  results  as  those  made  with  water  or  weak  alcohol. 
Alcohol  of  50  per  cent,  strength,  however,  may  be  said  to 
constitute  a  very  generally  desirable  medium.  The  sections 
must  be  very  thoroughly  stained  in  the  solution.  Asa  general 
rule  they  cannot  be  left  too  long  in  the  staining  fluid.  With 
the  powerful  solutions  obtained  with  anilin  a  few  minutes  or 
half  an  hour  will  frequently  suffice,  but  to  be  on  the  safe  side 
it  is  frequently  well  to  leave  the  sections  twelve  to  twenty- 
four  hours  in  the  fluid.  Up  to  a  certain  point  the  more  the 
tissues  are  stained  the  better  do  they  resist  the  washing-out 
process,  which  is  an  advantage.  For  researches  on  nuclei  it 

*  Historically  the  principle  of  this  method  is  due  to  HERMANN  and 
BOETTCHER;  but  it  is  generally  known  by  the  name  of  FLEMMING,  to 
whom  is  due  the  credit  of  having  greatly  improved  the  method  in  its  prac- 
tical details. 


ON   STAINING  WITH    COAL-TAR    COLOURS.  197 

is  said  that  the  solutions  made  with  anilin  had  better  be 
employed  only  with  preparations  well  fixed  in  chromo-aceto- 
osmic  acid,  as  the  basic  anilin  oil  may  easily  attack  chromatin 
if  not  specially  well  fixed. 

Material  fixed  in  chromo-osmic  mixtures  gives  a  sharper 
and  more  selective  stain  than  material  fixed  in  sublimate  or 
the  like.  During  the  staining  the  tissues  become  overittained, 
that  is  charged  with  colour  in  an  excessive  and  diffuse 
manner.  The  stain  must  therefore  now  be  differentiated  by 
removal  of  the  excess  of  colour. 

265.  Differentiation. — This  is  generally  done  with  alcohol, 
sometimes  pure,  sometimes  acidulated  (with  HC1).  The 
stained  sections,  if  loose  (celloidin  sections),  are  brought  into 
a  watch- glassful  of  alcohol ;  if  mounted  in  series  on  a  slide 
they  are  brought  into  a  tube  of  alcohol  (differentiation  can 
be  done  by  simply  pouring  alcohol  on  to  the  slide,  but  it  is 
better  to  use  a  tube  or  other  bath) .  It  is  in  either  case  well 
to  just  rinse  the  sections  in  water,  or  even  to  wash  them  well 
in  it,  before  bringing  them  into  alcohol. 

The  sections  in  the  watch  glass  are  seen  to  give  up  their 
colour  to  the  alcohol  in  clouds,  which  are  at  first  very  rapidly 
formed,  afterwards  more  slowly.  The  sections  on  the  slide 
are  seen,  if  the  slide  be  gently  lifted  above  the  surface  of  the 
alcohol,  to  be  giving  off  their  colour  in  the  shape  of  rivers 
running  down  the  glass.  In  a  short  time  the  formation  of 
the  clouds  or  of  the  rivers  is  seen  to  be  on  the  point  of 
ceasing  ;  the  sections  have  become  pale  and  somewhat  trans- 
parent, and  (in  the  case  of  chrom-osmium  objects)  have 
changed  colour,  owing  to  the  coming  into  view  of  the  general 
ground  colour  of  the  tissues,  from  which  the  stain  has  now 
been  removed.  (Thus  chrom-osmium- safraiiin  sections  turn 
from  an  opaque  red  to  a  delicate  purple.)  At  this  point  the 
differentiation  is  complete,  and  the  extraction  of  the  colour 
by  the  alcohol  must  be  stopped  instantly  (see  §  267). 

It  is  generally  directed  that  absolute  alcohol  be  taken  for 
differentiation.  This  may  be  well  in  some  cases,  but  in 
general  95  per  cent,  is  found  to  answer  perfectly  well. 

The  hydrochloric-acid-alcohol  process  had  better  only  be 
employed  with  tissues  well  fixed  with  "  Flemming,"  as  with 
tissues  imperfectly  fixed  it  may  cause  swellings.  Further, 


198  CHAPTER   XIV. 

the  acid  extracts  the  colour  mucli  more  quickly  from  resting 
nuclei  than  from  kinetic  nuclei,  which  is  an  advantage  or  a 
disadvantage  according  to  the  end  in  view. 

The  proportion  of  HC1  with  which  the  alcohol  should  be 
acidified  for  the  acid  process  should  be  about  1  :  1000,  or 
less ;  seldom  more. 

As  a  rough  and  ready  guide  to  the  beginner,  it  may  be 
stated  that  washing  out  should  be  done  with  neutral  alcohol 
whenever  it  is  desired  to  have  resting  nuclei  stained  as  well 
as  dividing  nuclei ;  the  other  processes  serving  chiefly  to 
differentiate  karyokinetic  figures. 

Differentiation  with  neutral  alcohol  is  known  as  "  neutral 
differentiation,"  or  "  neutral  extraction;"  and  differentia- 
tion with  hydrochloric  acid  is  known  as  "  acid  differentiation/' 
or  "acid  extraction." 

The  length  of  time  necessary  for  differentiating  to  the 
precise  degree  required  varies  considerably  with  the  nature 
of  the  tissues  and  the  details  of  the  process  employed ;  all 
that  can  be  said  is  that  it  generally  lies  between  thirty 
seconds  and  two  minutes.  The  acid  process  is  vastly  more 
rapid  than  the  neutral  process,  and  therefore  of  course  more 
risky. 

Other  differentiating  media  than  alcohol  and  hydrochloric 
acid  are  also  employed,  and  will  be  mentioned  in  their  proper 
places. 

266.  Substitution.— There  exists  a  mode  of  differentiation  that  is  both 
of  practical  importance  and  of  theoretical  interest — one  stain  may  be  made 
to  wash  out  another.  Thus  methylen  blue  and  gentian  violet  are  discharged 
from  tissues  by  aqueous  solution  of  vesuvin  or  of  eosin  ;  fuchsin  is  dis- 
charged from  tissues  by  aqueous  solution  of  methylen  blue.  The  second 
stain  "substitutes"  itself  for  the  first  in  the  general  "ground"  of  the 
tissues,  leaving,  if  the  operation  have  been  successfully  carried  out,  the 
nuclei  stained  with  the  first  stain,  the  second  forming  a  "contrast  "  stain. 

FLEMMING  differentiates  in  a  solution  of  Orange  G.  sections  that  have 
been  previously  stained  with  gentian  violet  (see  his  orange  method,  §  283). 
Flemming  attributes  the  differentiation  in  this  case  to  the  "  acid  "  qualities 
of  the  Orange.  I  am  not  able  to  say  how  far  the  "  acid  "  nature  of  dyes  in 
Ehrlich's  sense  confers  on  them  the  power  of  extracting  the  stains  of  basic 
colours,  or  of  less  acid  colours,  It  is  certain  at  any  rate  that  this  property 
is  also  possessed  by  some  basic  colours,  as  is  testified  by  two  of  the  examples 
given  above,  both  vesuvin  and  methylen  blue  being  basic  colours,  and  other 
examples  might  be  quoted. 

In  the  paper  of  EESEGOTTT  in  Zeit.f.  wiss.  Mi'k.,  v,  3,  1888,  p.  320,  it  is 


ON    STAINING    WITH    COAL-TAB   COLOURS.  199 

stated  as  a  very  general  rule  that  colours  that  do  not  give  a  nuclear  stain  by 
the  regressive  method  will  wash  out  those  that  do.  But  RESEGOTTI'S  expe- 
riments do  not  seem  to  me  to  constitute  a  case  in  point.  For  he  used  the 
second  colour,  if  I  understand  him  rightly,  in  alcoholic  solution ;  so  that  it 
remains  uncertain  how  far  the  differentiation  should  be  attributed  to  the 
second  colour  itself,  and  how  far  to  the  alcohol  used  as  a  vehicle.  The  same 
remark  applies  to  BENDA'S  Safranin-andvLichtgriin  process. 

267.  Clearing. — The  differentiation  having  been  carried  to 
a  satisfactory  point,  as  described  in  §  265,  the  extraction  of 
the  colour  may  be  stopped  by  putting  the  sections  into  water  ; 
but  the  general  practice  is  to  clear  and  mount  them  at  once. 

You  may  clear  with  clove  oil,  -ivhich  will  extract  some  more 
colour  from  the  tissues.  Or  you  may  clear  with  an  agent 
that  does  not  attack  the  stain  (cedar  oil,  bergamot  oil,  xylol, 
toluol,  etc.;  see  the  chapter  on  Clearing  Agents).  If  you 
have  used  neutral  alcohol  for  washing  out,  you  had  perhaps 
1  fetter  clear  with  clove  oil,  as  neutral  alcohol  does  not  always, 
if  the  staining  have  been  very  prolonged,  extract  the  colour 
perfectly  from  extra-nuclear  parts.  But  if  you  have  not 
stained  very  long,  and  if  you  have  used  acidulated  alcohol 
for  washing  out,  clove  oil  is  not  necessary,  and  it  may  be 
better  not  to  use  it,  as  it  somewhat  impairs  the  brilliancy  of 
the  stain.  A  special  property  of  clove  oil  is  that  it  helps  to 
differentiate  karyokinetic  figures,  as  it  decolmrs  resting  nuclei 
more  rapidly  than  these  in  division. 

Some  colours  are  much  more  sensible  to  the  action  of  clove 
oil  than  others ;  and  much  depends  on  the  quality  of  this 
much-adulterated  essence.  New  clove  oil  extracts  the  colour 
more  quickly  than  old. 

Series  of  sections  on  slides  are  conveniently  cleared  by 
pouring  the  clearing  agent  over  them. 

AVheii  the  clearing,  is  accomplished  to  your  satisfaction, 
either  mount  in  damar  or  balsam  ;  or,  stop  the  extraction  of 
the  colour,  if  clove  oil  have  been  used,  by  putting  the  sections 
into  some  medium  that  does  not  affect  the  stain  (xylol,  cedar 
oil,  etc.).  Chloroform  should  be  avoided,  either  as  a  clearer 
or  as  the  menstruum  for  the  mounting  medium. 

268.  General  Results. — The  results  depend  in  great  measure 
on  the  previous  treatment  of  the  tissues.      If  you  have  given 
them   a  prolonged    fixation   in  Flemrning's    stronfj    chromo- 


200  CHAPTER    XIV. 

aceto-osmic  mixture,  and  have  differentiated  after  staining 
with  acid  alcohol  and  cleared  with  clove  oil,  you  will  get, 
with  some  special  exceptions,,  nothing  stained  but  nucleoli 
and  the  chromatin  of  dividing  nuclei,  that  of  resting  nuclei 
remaining  unstained.  If  you  have  given  a  lighter  fixation, 
with  Flemming's  weak  mixture  or  some  other  fixing  agent 
not  specially  inimical  to  staining,  and  have  differentiated 
after  staining  with  neutral  alcohol,  you  will  get  the  chromatin 
of  resting  nuclei  stained  as  well. 

269.  HENNEGUY'S  Permanganate  Method  (Journ.  de  I'Anat.  et  de  la 
Physiol.,  xxvii,  1891,  p.  397). — This  method  is  based  on  the  fact,  discovered 
by  HENNEGUY,  that  permanganate  of   potassium  is  a  mordant  for  many 
anilin  dyes,  and  will  enable  a  good  stain  to  be  procured  in  cases  in  which 
the  usual  methods  fail. 

Sections  are  treated  for  five  minutes  with  1  per  cent,  solution  of  perman- 
ganate of  potassium.  They  are  then  washed  with  water  and  stained  (for 
about  half  the  time  that  would  have  been  taken  if  they  had  not  been  mor- 
danted with  the  permanganate)  in  safranin,  rubin,  gentian  violet,  vesuvin, 
or  the  like.  The  stain  that  succeeds  the  best  is  a  safranin  solution  prepared 
with  anilin  water  and  absolute  alcohol  (see  below,  §  272).  After  staining 
they  are  differentiated  with  alcohol,  followed  by  clove  oil  in  the  usual  way. 
The  progress  of  the  decoloration  should  be  controlled  under  the  microscope, 
in  order  that  it  may  be  stopped  at  the  proper  moment.  It  goes  on  in  general 
slowly,  and  the  slower  it  proceeds  the  more  selective  will  be  the  resultant 
stain.  The  decoloration  sometimes  continues  even  after  the  sections  have 
been  mounted  in  balsam,  especially  if  all  traces  of  clove  oil  have  not  been 
removed  before  mounting.  It  may  thus  happen  that  preparations  which  are 
insufficiently  washed  out  at  the  moment  of  mounting  show  a  perfectly 
differentiated  stain  twenty-four  or  forty-eight  hours  afterwards.  The  stain 
is  either  purely  nuclear,  or  in  part  plasmatic,  according  to  the  extent  of  the 
differentiation.  I  consider  that  it  may  occasionally  be  useful. 

The  mordanting  action  of  permanganate  of  potassium  on  anilin  stains  is 
so  energetic  that  if  it  have  been  overmuch  prolonged  before  staining  with 
safranin,  or,  still  more,  with  rubin,  it  becomes  almost  impossible  to  wash 
out  the  sections  properly ;  it  may  be  necessary  to  leave  them  for  a  month  or 
more  in  clove  oil. 

270.  OHLMACHER'S  Formaldehyde  Process  (Medical  News,  February 
16th,  1895).— Ohlmacher  states  that  formaldehyde  is  a  powerful  mordant 
for  tar  colours.     Tissues  may  either  be  mordanted  separately  by  treatment 
for  a  short  time  (one  minute  is  enough  for  cover-glass  preparations)  with  a 
2  per  cent,  to  4  per  cent,  formalin  solution;  or  the  formalin  may  be  com- 
bined with  the  stain.    One  gramme  of  fuchsin  dissolved  in  10  c.c.  of  absolute 
alcohol  may  be  added  to  100  c.c.  of  4  per  cent,  formalin  solution.     Or  satu- 
rated alcoholic  solution  of  gentian  violet,  or  methyl  violet  5  B,  may  be  added 
to  4  per  cent,  formalin  solution  in  the  proportion  of  1  :  10.     Or  formalin- 


ON    STAINING    WITH    GOAL-TAB    COLOURS.  201 

roethylen  blue  may  be  made  by  dissolving  1  grm.  of  methylen  blue  in  100 
c.c.  of  the  formalin  solution.  Sections  are  said  to  stain  in  half  a  minute, 
and  to  resist  alcohol  muc*h  more  than  is  the  case  with  those  treated  by  the 
usual  solutions.  The  formalin  solution  of  safranin  (Safranin  0,  from 
Grubler)  is  said  to  give  a  plasma  stain  behaving  in  all  particulars  like  eosin. 

271.  Choice  of  a  Stain. — One  might  think  that  it  would  be 
quite  sufficient  for  all  practical  purposes  to  possess  one  good 
red  stain  and  one  good  blue  one,  so  that,  for  instance, 
safranin  and  gentian  violet  should  be  sufficient  for  the  most 
exacting  of  laboratories.  But  I  think  that  for  delicate  work, 
at  any  rate,  it  is  desirable  to  possess  one  or  two  more.  We 
have  to  take  account  of  the  manner  in  which  these  colours 
behave  when  used  in  combination  with  the  plasma  stains  that 
it  may  be  desired  to  employ.  And  there  is  another  point 
that  is  not  undeserving  of  attention.  Some  of  the  dyes  dis- 
cussed in  the  following  chapter  give  a  stain  of  a  somewhat 
dead  or  dull  quality,  so  much  so  that  chromosomes  and 
nucleoli  frequently  come  out  quite  opaque.  Gentian  violet  is 
in  this  case  ;  whilst  dahlia,  which  is  otherwise  near  to  it  in 
hue,  is  not.  Safranin  and  methyl  green,  on  the  other  hand, 
leave  the  structures  beautifully  transparent.  This  is  an  ad- 
vantage with  thick  sections,  and  sometimes  for  other  reasons  ; 
but  this  transparency  of  the  elements  is  unfortunately 
favourable  to  the  production  of  diffraction  lines,  which  may 
be  a  hindrance  to  good  definition  in  delicate  work.  So  that 
the  dead  colours,  such  as  gentian,  have  a  certain  advantage 
for  work  with  very  thin  sections  and  where  very  fine  defini- 
tion of  chromatiii  is  required;  whilst  the  transparent  or 
semi-transparent  colours,  such  as  safranin,  should  be  pre- 
ferred for  thick  sections.  I  would  also  add  that  it  always 
seems  to  me  that  the  blue  stains,  such  as  gentian,  are  less 
favourable  for  work  with  artificial  light.  They  give  more 
or  less  dichroic  images,  which  are  not  favourable  to  good 
definition. 

To  sum  up,  I  would  recommend  safranin  for  a  red  chro- 
matin  stain,  and  gentian  for  a  blue  one,  except  where 
special  conditions  suggest  another  choice. 


CHAPTER  XY. 

THE    COAL-TAR    CHROMATIS"    STAIXS. 
A.  Regressive  Stains. 

272.  Safranin. — One  of  the  most  important  of  these  stains , 
on  account  of  its  great  power,,  brilliancy,  and  superior  per- 
manence in  balsam,  and  also  on  account  of  the  divers  degrees 
of  electivity  that  it  displays  for  the  nuclei  and  other  consti- 
tuent elements  of  different  tissues. 

The  great  secret  of  staining  with  safranin  is  to  get  a  good 
safranin.  It  is  needful  here  to  insist  most  urgently  on  what 
was  said  above  (§  204).  Before  thinking  of  working  with 
this  important  reagent  you  should  go  to  Gr abler  &  Hoi  1  born 
or  to  Miinder,  and  order  the  safranin  you  want,  specifying 
whether  you  want  it  for  staining  nuclei  or  for  staining  elastic 
fibres,  or  for  what  other  purpose  you  may  require  it. 

There  are  presumably  at  least  a  score  of  sorts  of  safranin  in  the  market, 
differing  to  a  considerable  extent  in  colour,  weight,  solubility,  and  histo- 
logical  action.  Some  are  easily  soluble  in  \vater  and  not  so  in  alcohol,  some 
the  reverse,  and  some  freely  soluble  in  both.  Fourteen  brands,  supplied  by 
Griibler  and  by  Miinder,  have  been  studied  by  RESEGOTTI  (Zeit.  f.wiss. 
Mile.,  v,  3,  1888,  p.  320).  Resegotti  obtained  his  best  results  with  the 
brands  "  Safranin  wasserloslich,"  "  Safranin  spiritusloslich,"  "  XX," 
"  XXBN,"  "TB,"  furnished  by  Griibler,  and  with  the  brands  "  Rein,:>  "  0," 
"  FII,"  and  "Cone.,"  supplied  by  Miinder. 

The  brand  I  have  been  using  for  a  long  time,  and  which 
gives  good  results,  is  the  "  Safraniii  0  "  of  Griibler  &  Co. 
It  should  be  remembered  that  as  the  processes  of  manufacture 
are  constantly  changing,  the  properties  of  the  products  are 
sure  to  vary  somewhat  from  time  to  time. 

Staining. — The  majority  of^afranins  are   not    sufficiently 


COAL-TAK   CHKOMATJN    STAINS.  203 

soluble  in  water,  so  that  solutions  in  other  menstrua  must  be 
employed. 

A  solution  much  used  some  time  ago  is  that  of  PFITZNER 
(Morph.  Jalrb.,  vi,  p.  478,  and  vii,  p.  291),  composed  of  saf- 
ranin  1  part,  absolute  alcohol  100  parts,  and  water  200  parts, 
the  last  to  be  added  only  after  a  few  days. 

The  solution  of  FLEMMING  (Arch.f.  mik.  Anut.,  xix,  1881, 
p.  317)  is  a  concentrated  solution  in  absolute  alcohol,  diluted 
with  about  one  half  of  water. 

The  solutions  of  BABKS  (Arch.  /.  mik.  Anat.,  1883,  p.  356) 
are  (A)  a  mixture  of  equal  parts  of  concentrated  alcoholic 
solution  and  concentrated  aqueous  solution  (this  is  very  much 
to  be  recommended) ,  and  (B)  a  concentrated  or  supersaturated 
aqueous  solution  made  with  the  aid  of  heat. 

Some  people  still  employ  simple  aqueous  solutions. 

The  anilin  solution  of  BABES  (Zeif.  f.  wiss.  Mik.,  iv,  4,. 
1887,  p.  470)  consists  of  water  100  parts,  anilin  oil  2  parts, 
and  an  excess  of  safraniii.  The  mixture  should  be  warmed 
to  from  60°  to  80°  C.,  and  filtered  through  a  wet  filter.  This 
solution  will  keep  for  a  month  or  two. 

ZWAAKDKMAKER  (Zeit.  f.  u'l'stf.  Mik.,  iv,  2,  1887,  p.  212) 
makes  a  mixture  of  about  equal  parts  of  alcoholic  safraniii 
solution  and  anilin  water  (saturated  solution  of  aiiiliii  oil  in 
water ; — to  make  it,,  shake  up  "  anilin  oil;"  which  is  nothing 
but  pure  anilin,  with  water,  and  filter).  This,  I  find,  will 
keep  for  many  months,  perhaps  indefinitely. 

I  myself  use  equal  parts  of  saturated  solution  in  anilin 
water,  and  saturated  solution  in  absolute  alcohol. 

Any  of  these  stains  may  be  used  with  any  of  the  following 
differentiation  processes.  Of  course  you  will  have  to  stain 
longer  in  the  weaker  solutions.  As  to  the  anilin  solutions 
see  ante,  §  264. 

Differentiation. — For  general  directions  for  differentiation 
and  clearing  see  above,  §§  265  and  267. 

Neutral  differentiation  with  pure  alcohol,  followed  by 
clove  oil  gives  resting  chromatin  stained,  as  well  as  kinetic 
chromatin. 

FLOIM  ING'S  <//•/'//  differentiation  (Zeif.  /.  /'•/>•*.  Mil:.,  i,  3. 
1884,  p.  350). — Differentiate,  until  hardly  any  more  colour 
comes  awav,  in  alcohol  acidulated  with  about  0*5  per  cent, 
of  hydrochloric  acid,  followed  by  pure  alcohol  and  clove  oil. 


204  CHAPTER  XV. 

(You  may  use  the  HC1  in  watery  solution  if  you  prefer  it.) 
Or  you  may  use  a  lower  strength,  viz.  0*1  per  cent,  at  most 
(see  Arch./,  mik.  Anat.,  xxxvii,  1891,  p.  249)  ;  and  this  I 
find  is  generally  preferable. 

Objects  are  supposed  to  have  been  well  fixed — twelve 
hours  at  least — in  the  strong  chromo-aceto-osmic  mixture, 
and  stained  for  some  hours.  In  this  way  you  get  kinetic 
chromatin  and  nucleoli  alone  stained  (if  the  fixation  have 
been  performed  as  above  directed). 

PODWYSSOZKI  (Beitr.  z.  path.  Anat.,  i,  1886  ;  Zeit.  f.  u'iss. 
Mile.,  iii,  3,  1886,  p.  405)  differentiates  (for  from  a  few 
seconds  to  two  minutes)  in  a  strongly  alcoholic  solution  of 
picric  acid,  followed  by  pure  alcohol.  Same  results  (except 
that  the  stain  will  be  brownish  instead  of  pure  red) . 

BABES  recommends,  for  sections  stained  in  the  anilin  solu- 
tion, treatment  with  iodine,  according  to  the  method  of  GRAM 
(see  next  section).  This  process  has  also  been  recommended 
by  PRENANT  (Int.  Monatsschr.  /.  Anat.,  etc.,  iv,  1887,  p. 
368). 

It  has  been  shown  by  OHLMACHER  (Journ.  Amer.  Med.  Ass.,  vol.  xx, 
No,  5,  Feb.  4,  1893,  p.  Ill)  that  if  tissues  be  treated  with  solutions  con- 
taining iodine  or  picric  acid  after  staining  with  safranin,  there  may  be 
produced  in  the  tissue  elements  a  precipitate  of  a  dark  red  substance  of  a 
crystalline  nature,  but  of  lanceolate,  semilunar,  falciform,  or  navicellar 
forms.  This  precipitate  is  formed  both  in  normal  and  pathological  tissue, 
readily  i*1  carcinomatous  tissues  ;  and  Ohlinacher  concludes  that  many  of  the 
bodies  that  have  been  described  as  "  coccidia,"  "  sporozoa,"  or  other 
<c  parasites  "  of  carcinoma  are  nothing  but  particles  of  this  precipitate. 

See  also  the  differentiation  process  of  MAETINOTTI  and  RESEGOTTI  {Zeit. 
/.  wiss.  Mik.,  iv,  3,  1887,  p.  328)  for  alcohol-fixed  material ;  and  of  GARBINI 
(Zeit.  f.  wiss.  Mik.y  v,  2,  1888,  p.  170. 

In  preparations  made  with  chromo-aceto-osmic  acid, 
safranin  stains,  besides  nuclei,  elastic  fibres,  the  cell  bodies 
of  certain  horny  epithelia,  and  the  contents  of  certain  gland- 
cells  (mucin,  under  certain  imperfectly  ascertained  condi- 
tions) . 

The  stain  is  perfectly  permanent. 

273.  Gentian  Violet.— One  of  the  best  of  these  stains.  It 
may  be  used  in  aqueous  solution,  or  in  alcoholic  solution 
diluted  with  about  one  half  of  water  (FLEMMING,  Zells.  Kern, 
u.  Zellth.,  1882,  p.  384),  and  the  stain  may  be  differentiated 


COAL-TAR    CHROMATIN   STAINS.  205 

with  neutral  alcohol,  or  (FLEMMING,  Zeit.  f.  wiss.  Mik.,  1, 
1884,  p.  350)  acidulated  alcohol,  as  directed  for  safranin. 
BIZZOZERO  (Zeit.  f.  iciss.  Mik.,  iii,  1,  1886,  p.  24)  stains  in  a 
solution  borrowed  from  that  of  EHRLICH  for  bacteria,  and 
consisting  of — 

Gentian  violet      .  ...      1  part. 

Alcohol        .  .  .  .  .15  parts. 

Anilin  oil    .  .  .  .  3      „ 

Water 80      „ 

The  complicated  chromic-acid  differentiation  process  recom- 
mended by  him  appears  to  me  quite  superfluous. 

In  some  cases  it  may  be  useful  to  employ  the  method 
devised  by  GRAM  for  the  differentiation  of  bacteria  in  tissues 
(Fortschr.  d.  Medicin.,  ii,  1884,  No.  6  ;  British  Med.  Journ., 
Sept,  6th,  1884,  p.  486;  Journ.  Roy.  Hie.  Soc.  [N.S.],  iv, 
1884,  p.  817). 

In  Gram's  method  the  sections  ar£  treated,  after  staining, 
with  a  solution  composed  of — 

Iodine  .....  1  gramme, 

Iodide  of  potassium  .  .  2  grammes, 

Water  ....       300          „ 

for  two  or  three  minutes,  until  they  become  black.  They 
are  then  differentiated  with  neutral  alcohol,  until  they  turn 
grey,  and  are  then  finally  differentiated  with  clove  oil. 

By  this  process,  in  resting  nuclei  the  nucleoli  alone  are 
stained,  or  the  chromatin  if  stained  is  pale  ;  in  dividing 
nuclei  the  chromatin  is  stained  with  great  intensity,  being 
nearly  black  in  the  equatorial  stage. 

Gentian  violet  is  an  exceedingly  powerful  stain,  quite  as 
precise  as  safranin,  to  which  it  is  perhaps  even  preferable  for 
much  work  with  very  thin  sections  (thick  sections  with 
closely  packed  nuclei  may  easily  come  out  too  dark).  It 
lends  itself  well  to  double-staining  with  red  or  yellow  plasma 
stains. 

The  stain  keeps  well  if  the  preparations  be  not  unduly 
exposed  to  light. 

Gentian  violet  in  acid  solution  stains  the  nuclei  of  fresh  tissues,  and  dis- 
solved in  indifferent  media  is  sometimes  very  useful  for  staining  intra  vitam 
(see  §  201). 

HERMANN  (Arch.  mik.  Anat.,  xxxiv,  1889,  p.  58)  first  stains  for  twenty- 
four  hours  or  more  in  safranin,  differentiates  incompletely  with  .alcohol, 


206  CHAPTER   XV. 

then  stains  for  three  to  five  minutes  in  the  anilin-water  gentian  solution, 
treats  with  the  iodine  solution  for  one  to  three  hours,  and  finally  differentiates 
with  alcohol. 

274.  Thionin. — The  hydrochloride  of    thionin,   or  violet  of 
Lauth,  is  a  colour  chemically  nearly  allied  to  methylen  blue. 
It   may   be    obtained    from  'Griibler  &  Hollborn.       I    have 
classed   it    here    as   a  'regressive   stain,  but  its  action   is  so 
selective  from  the  first  that   it  may  almost  be  considered  to 
be  a  progressive  stain. '    If  you  stain  for  "only  a  short  time  (a 
few  minutes)  in  a  concentrated  aqueous  solution,  hardly  any- 
thing but  the  chromatin  will  be  found  to  be  stained.      If  the 
staining  be  prolonged,  plasmatic  elements  will  begin  to  take 
up  the  colour.      After  a  short  stain  no  special  differentiation 
is   required ;   all    that  is  necessary   is    to    rinse  with   water, 
dehydrate,  and  mount.      After  a  strong  stain  you  differentiate 
with  alcohol  in  the  usual  way,  with  this  advantage,  that  the 
:stain  is  so  highly  resistent  to   alcohol  that  there  is  no  risk 
whatever  of   overshooting  the    mark  ;  the   stain  will   not    be 
more  extracted  in  an"  hour"  than  "that  of  gentian  or  dahlia  is 
in  a  minute,  so  that  the  process  may  be  controlled  under  the 
microscope  if  desired.      For  this  reason   I   think  this    stain 
may  be  useful  to  beginners,  but  I  myself  prefer  gentian.      It 
is  a  very  powerful  stain. 

Thionin  is  a  specific  stain  for  mucin,  q.  v.  Some  observers  have  found 
the  stain  to  fade.  WOLFF  (Zeit.f.  wiss.  Mik.,  xv,  3, 1899,  p.  312)  says  that, 
to  avoid  this,  preparations  should  be  mounted  in  a  little  solid  colophonium 
or  balsam  melted  over  a  flame. 

275.  Other  Regressive  Stains— The  foregoing,  I  think,  may 
suffice  for  most  practical  purposes,  but  the  following  may  be 
mentioned. 

Dahlia  (FLEMMING,  Arch.  f.  mik.  Anat.,  xix,  1881,  p. 
317). — The  stain  is  paler  in  the  nuclei  than  with  gentian  or 
safranin.  The  cytoplasmic  granulations  of  certain  cells  are 
sharply  stained. 

Dahlia  is  also  a  useful  nuclear  stain  for  fresh  tissues  (v.  EHRLICH,  Arch, 
f.  mil:.  Anat.,  xiii,  1876,  p.  263).  For  these  the  aqueous  solution  must  be 
acidulated  with  (7'5  per  cent.)  acetic  acid  ;  or  you  may  stain  in  a  neutral 
solution,  and  wash  out  with  acidulated  water. 

Victoria  Blue  (Victoriablau)  (LUSTGABTEN,  Med.  Jahrb.  k. 
Ges.  d.  Aerzte  zu  Wien,  1886,  pp.  285—291).  « 


COAL-TAR   CHROMATIN    STAINS.  207 

Victoria  has  a  special  affinity  for  elastic  fibres.  For  this 
object  Lustgarten  recommends  an  alcoholic  solution  of  the 
dye  diluted  with  two  to  four  parts  of  water.  Fixation  in 
chrom-osmium,  or  at  least  in  a  chromic  mixture,  is,  I  believe, 
a  necessary  condition  to  this  reaction.  And  you  must  stain 
for  a  long  time. 

Victoria  has  also  a  special  affinity  for  mucus- cells,  from 
which  it  is  not  washed  out  by  alcohol. 

MAGDALA  RED  (NAPTHALIN  RED,  ROSE  DE  NAPTHALINE). 

Fuchsin  (meaning  the  basic  fuchsins,  a  series  of  Rosanilin  salts  having 
very  similar  reactions,  and  found  in  commerce  under  the  names  of  FUCHSIN. 
ANILIX  RED,  RUBIN,  ROSEIN,  MAGENTA,  SOLFERINO,  CORALLIN). — GRASER 
(Deutsche  Zeit.  f.  Chirurgie,  xxvii,  1888,  pp.  538—584;  Zeit.  f.  wiss.  Mil;., 
v,  3, 1888,  p.  378)  stains  for  twelve  to  twenty-four  hours  in  a  dilute  aqueous 
solution,  washes  out  for  a  short  time  in  alcohol,  stains  for  a  few  minutes  in 
aqueous  solution  of  inethylen  blue,  and  dehydrates  with  alcohol.  A  double 
stain.  Chromatin  and  nucleoli  red,  all  the  rest  blue. 

ZIEHI/S  Carbolic  Fuchsin  (from  Zeit.  f.  iriss  Mik.,  vii,  1, 
1890,  p.  39). 

The  stain  is  made  either  by  taking — 

Fuchsin        ....         1  gramme, 
Acid,  carbol.  crist.       .          .         5  grammes, 
Alcohol        ....        10         „ 

Aq.  dest 100 

or  by  saturating  a  5  per  cent,  aqueous  solution  of  carbolic 
acid  with  concentrated  alcoholic  solution  of  fuchsin  (the 
saturation  of  the  carbolic  solution  with  fuchsin  is  made  mani- 
fest by  the  formation  of  .a  metallic -looking  pellicle  on  the 
surface  of  the  liquid) .  The  stain  is  washed  out  with  alcohol 
followed  by  clove  oil. 

Bismark  Brown  has  this  advantage,  that  being  sufficiently  resistent  to 
alcohol  it  may  be  utilised  for  staining  entire  objects. 

KAISER  (Biblioth.  Zool.  H.  7,  1  Halfte,  1891  ;  Zeit.  f.  wiss.  Mik.,  viii, 
3,  1891,  p.  363)  stains  for  forty-eight  hours,  and  at  a  temperature  of  60  C. 
in  saturated  solution  of  Bismarck  brown  in  60  per  cent,  alcohol  (the  solution 
to  be  made  in  boiling  alcohol),  and  washes  out  (until  all  is  decoloured  except 
the  karyokinetic  figures)  in  60  per  cent,  alcohol,  containing  2  per  cent, 
hydrochloric  acid  or  3  per  cent,  acetic  acid. 
METHYL  VIOLET. 

BENZOAZURIN  (MARTIN,  Zeit.f.  wiss.  Mik.,  vi,  3,  1889,  p.  193).     Stain 
for  an  hour  or  so  in  dilute  aqueous  solution,  and  wash  out  with  HC1  alcohol. 
METHYLEN  BLUE. 

Nigrosin  (£RRERA  Proc.-Verb.  Soc.  Beige  de  Mic.,  1881,  p.  134).  The 
stain  resisjts  alcohol  well. 


208  CHAPTER  XV. 

With  Toluidin  Blue  I  have  had  some  superb  stains  of  chromatin,  un- 
fortunately accompanied  by  a  diffuse  staining  of  cytoplasm. 

MANN  (Zeit.  /.  wiss.  Mik.,  xi,  4,  1894,  p.  489)  states  that  he  has  had 
good  results  by  staining  with  it  after  eosin,thus  obtaining  a  double  stain. 

See  further  as  to  the  micro-chemical  properties  of  this  dye,  HAERIS,  The 
Philadelphia  Medical  Journal,  May  14th,  1898.  It  much  resembles 
rnethylen  blue. 


JB.  Progressive  Stains. 

276.  As  regards  the  progressive  nuclear  stains,  the  reader  is 
reminded  that  many,  if  not  most,  of  the  basic  coal-tar  colours  give  a  nuclear 
stain  of  greater  or  less  purity  if  they  are  used  in  solutions  acidified  with 
acetic  acid.  Under  the  present  heading,  only  those  are  mentioned  which  give 
in  all  respect,  alike  as  regards  precision  and  permanence,  simplicity  of  mani- 
pulation and  other  qualities,  a  really  valuable  stain. 

277.  Methyl  Green. — This  is  the  most  common  in  commerce 
of  the  "Aiilin"  greens.  It  appears  to  go  by  the  synonyms 
of  Methylanilin  green,  Grilnpulver,  Vert  Lumiere,  Lichtgrun  ; 
these  two  last  are  in  reality  the  name  of  another  colour. 
When  first  studied  by  Calberla,  in  1874  (Morphol.  Jahrb.,  in, 
1887,  p.  625),  it  went  by  the  name  of  Vert  en  cristaux.  It 
is  commonly  met  with  in  commerce  under  the  name  of  more 
costly  greens,  especially  under  that  of  Iodine  green.  It  is 
important  not  to  confuse  it  with  the  latter,  nor  with  Aldehyde 
green  (Vert  d'Eusebe),  nor  with  the  phenylated  rosaiiilins, 
Paris  green,  and  Vert  d'AIcali,  or  Veridine. 

Methyl  green  is  the  chloromethylate  of  zinc  and  penta- 
methyl-rosanilin- violet.  It  is  obtained  by  the  action  of 
methyl  chloride  on  methyl  violet.  The  commercial  dye 
always  contains  unconverted  methyl  violet  as  a  consequence 
of  defective  purification.  It  is  sometimes  adulterated  with 
anilin  blue  (soluble  blue) .  It  is  also  sometimes  adulterated 
with  a  green  bye-product  of  the  manufacture, — the  chloride 
of  nona-methyl-para-leukanilin  (see  BENEDIKT  and  KNECHT'S 
Chemistry  of  the  Coal-tar  Colours). 

MAYEE  (Mitth.  Zool.  8 tat.  Neapel,  xii,  1896,  p.  312)  says  that  the  presence 
of  the  blue  impurity  can  be  demonstrated  by  placing  a  drop  of  the  solution 
of  the  ^dye  on  filter  paper,  and  holding  the  green  spot  over  a  bottle  of 
ammonia.  If  the  methyl  green  is  pure,  the  spot  will  disappear ;  if  not  pure, 
it  will  turn  violet.  He  also  says  that  the  violet  can  be  easily  removed  by 


COAL-TAR    CHROMATIN    STAINS.  209 

agitating  the  aqueous  solution  with  chloroform.  FISCHER  (Fixirung, 
Fdrbung,  u.  Bau  d.  Protoplasmus,  p.  89)  shakes  up  the  solution  in  a  burette 
with  a  little  amyl  alcohol,  which  quickly  becomes  violet  and  collects  at  the 
top,  whence  it  may  be  decanted  off  and  the  operation  repeated. 

Methyl  green  is  extremely  sensitive  to  the  action  of  alkalies. 
It  is  therefore  important  to  use  it  only  in  acidified  solutions, 
and  to  use  only  acid,  or  at  least  perfectly  neutral  fluids  for 
washing  and  mounting. 

This  is  an  extremely  important  histological  reagent.  Its 
chief  use  is  as  a  chromatin  stain  for  fresh,  unfixed  tissues. 
For  this  purpose  it  should  be  used  in  the  form  of  a  strong 
aqueous  solution  containing  a  little  acetic  acid  (about  1  per 
cent,  in  general).  Tfie  solutions  must  always  be  acid.  (If 
the  tissues  have  been-  previously  fixed  with  acetic  acid  you 
will  not  get  a  chromatin  stain.  The  same  applies  to  fixation 
with  acetic  acid  sublimate  :  whilst  pure  sublimate  will  allow 
of  a  chromatin  stain  (BUKCKHAKDT,  La  Cellule,  xii,  1897, 
p.  364).  You  may  wash  out  with  water  (best  Aidulated) 
and  mount  in  some  acid  aqueous  medium  containing  a  little 
of  the  methyl  green  in  solution.  The  mounting  medium,  if 
aqueous,  must  be  acidulated. 

Employed  in  this  way,  with  fresh,  unfixed  tissues,  methyl 
green  is  a  pure  chromatin  stain,  in  the  sense  of  being  a 
precise  colour  reagent  for  chromatin.  For  in  the  nucleus  it 
stains  nothing  but  chromosomes,  or  chromatin  elements  :  it 
does  not  stain  plasmatic  nucleoli,  nor  caryoplasm,  nor  achro- 
matic filaments.  Outside  the  nucleus  it  stains  some  kinds  of 
cytoplasm  and  some  kinds  of  formed  material,  especially 
glandular  secretions  (silk,  for  instance,  and  mucin).  The 
chromatin  elements  are  invariably  stained  of  a  bright  green 
(with  the  exception  of  the  nuclein  of  the  head  of  some 
spermatozoa),  whilst  extra-nuclear  structures  are  in  general 
stained  in  tones  of  blue  or  violet.  But  this  metachromatic 
reaction  is  probably  due  to  the  methyl-violet  impurity,  and 
is  not  obtained  with  a  chemically  pure  methyl  green. 

Besides  being  a  perfectly  precise  test  for  chromatin  in  the 
fresh  nucleus,  methyl  green  has  other  advantages.  Staining 
is  instantaneous ;  overstaining  never  occurs.  The  solution 
is  very  penetrating,  kills  cells  instantly  without  swelling  or 
other  change  of  form,  and  preserves  their  forms  for  at  least 
some  hours,  so  that  it  may  be  considered  as  a  delicate  fixa- 

14 


210  .  CHAPTER   XV. 

tive.  It  may  be  combined  without  precipitating  with  divers 
fixing  or  preserving  agents,  ^smic  acid  (0*1  to  1  per  cent.) 
may  be  added  to  it,  or  it  may  be  combined  with  solution  o£ 
EIPART  and  PETIT  (this,  by  the  way,  is  an  excellent  medium 
for  washing  out  in  and  mounting  in). 

Alcoholic  solutions  may  also  be  used  for  staining.  They 
also  should  be  acidulated  ivith  acetic  acid. 
'  The  stain  does  not  keep  easily.  It  is  difficult  to  mount  it 
satisfactorily  in  balsam,  because  the  colour  does  not  resist 
alcohol  sufficiently  (unless  this  be  charged  with  the  colour). 
The  resistance  of  the  colour  to  alcohol  is,  however  (at  all 
events  if  it  be  used  in  the  EHRLICH-BIONDI  combination),  con- 
siderably increased  by  treating  the  sections  for  a  few  minutes 
with  tincture  of  iodine  before  staining  (M.  HEIDENHAIN). 
And  SQUIEE  declares  that  thorough  washing  with  water 
before  passing  into  the  alcohol  has  the  *same  effect. 

Of  preparations  mounted  with  excess  of  colour  in  the  usual 
aqueous  lAdia  the  most  fortunate  only  survive  for  a  few 
months.  Dr.  HENNEGUY,  however,  writes  to  me  that  it  keeps 
well  in  B RUN'S  glucose  medium. 

Undoubtedly  methyl  green  is  one  of  the  most  valuable 
stains  yet  known.  It  is  the  classical  cJtromatin  stain,  for  fresh 
tissues. 

It  was  first  pointed  out,  I  believe,  by  HESCHL  (Wiener  med.  Wochenschr., 
2,  1879),  that  methyl  green  is  a  reagent  for'amyloid  degeneration.  His 
observations  were  confirmed  by  CUKSCHMAJSTN  (Virchow's  Arch.,  vol.  Ixxix, 
1880,  p.  556),  who  showed  that  it  colours  amyloid  substance  of  an  intense 
violet;  but  this  (as  pointed  out  by  SQUIEE,  Methods  and  Formulse,  etc., 
Churchill,  1892;  p.  37)  is  undoubtedly  due  to  its  containing  methyl  violet 
as  an  impurity. 

278.  Bismarck  Brown  (Manchester  Brown,  Phenylen  Brown, 
Vesuvin,  La  Phenicienne) . — A  fairly  pure  nuclear  stain  that 
will  work  either  with  fresh  tissues  or  with  such  as  have  been 
hardened  in  chromic  acid. 

The  colour  is  not  very  easily  soluble  in  water.  You  may 
boil  it  in  water,  and  filter  after  a  day  or  two  (WEIGERT,  in 
Arch.  /,  mik.Anat.,  xv,  1878,  p.  258).  You  may  add  a  little 
acetic  or  osmic  add  to  the  solution.  ^AYSEL  (ibid.,  xviii, 
1880,  pp.  237,  250)  dissolves  the  co-lour  in  acetic  acid  (this 
solution  does  not  give  a  permanent  stain).  Alcoholic  solu- 


COAL-TAK    CHROMATIN    STAINS.  211 

tions  may  also  be  used,  e.  g.  saturated  aqueous  solution 
diluted*  with  one  third  volume  of  90  per -cent,  alcohol;  or 
CALBERLA'S  glycerin-aiid-alcohol  mixture,  or  dilute  glycerin 
.  (say  of  40  per  cent,  to  50  per  cent.)  may  very  advan- 
tageously be  employed. 

The  watery  solutions  must  be  frequently  filtered.  The 
addition  to  them  of  carbolic  'acid  has  been  recommended 
(vide  Journ.  Roy.  Hie.  8oc.,  1886,  p.  908).  MAYER,  however 
(Mitth.  Zool.  Stat.  Neapel,  xii,  1896,  p.  315),  points  out  that 
frequent  filtering  has  the  disadvantage  that  the  paper 
absorbs  a  great  deal  of  the  colour.  Bismarck  brown  stains 
rapidly,  but  never  overstains.  The  stain  is  permanent  both 
in  balsam  and  in  glycerin. 

The  chief  use  of  this  eolour  is  for  staining  objects  in  toto; 
but  it  may  be  employed  for  staining  sections  by  the  regres- 
sive method  (§  275),  and  also  for  intra-vitam  staining  (§  201) 
(for  this  purpose  it  is  necessary  to  see  that  the  colour 
-employed  be  pure  and  neutral).  9 

279.  Methyl  Violet   (Methylanilin  Violet,  Anilin  Violet,  Paris 

Violet).— GEASER  (Deutsche  Zeit.f.  Chirurgie,xxv\\,  1888,  pp.  538— 584; 
Zeit.f.  wiss.  Mik.,  v,  3,  1888,  p.  378)  recommends  the  following  process j 

Sections  are  stained  for  from  twelve  to  twenty-four  hours  in  a  (presum- 
ably aqueous)  solution  so  dilute  that  at  the  end  of  that  time  the  sections 
will  have  taken  up  all  the  colour  from  the  liquid.  They  are  then  washed 
out  for  a  short  time  in  acidulated  alcohol,  and  then  in  pure  alcohol  (followed 
presumably  by  clearing  and  mounting  in  balsam).  SCHIEFFEBDECKEB,  whose 
account  is  here  quoted,  says  that  the  results,  as  regards  nuclear  figures,  are 
even  finer  than  with  safrafiin.  The  method  is  applicable  to  objects  fixed  in 
"  Flemming." 

A  useful  stain  for  fresh  tissues  is  also  obtained  by  using  dilute  acetic  acid 
in  the  manner  recommended  above  (by  EHBLICH)  for  Dahlia  (§  275). 


CHAPTER  XVI. 

THE    COAL-TAE  PLASMA    STAINS. 

280.  As  to  Plasma  Stains. — By  a  plasma  stain  is  generally 
meant,  rather  vaguely,  one  that  stains  the  extra-nuclear  parts 
of  cells  and  the  formed  material  of  tissues,  or  one  of  these. 
To   be  precise  the   class   ought  to   be    subdivided,  and   we 
ought  to  speak  of  cytoplasm  stains,  granule  stains,  grouiid- 
substance  stains,  or  the  like.      But  the  vague  general  sense 
of  the  term  will  be  sufficient  for  the  purposes  of  the  present 
chapter. 

Good  plasma  stains  are  much  wanted.  Unfortunately 
such  a  thing  can  hardly  be  said  to  exist ;  for  it  is  not  enough 
to  require  of  a  plasma  stain  that  it  should  stain  extra-nuclear 
material.  It  is  also  desirable  that  it  should  do  so  in  as 
selective  a  way  as  possible.  Now  all  plasma  stains  are 
more  or  less  diffuse  stains.  Many  exhibit  considerable 
selectivity,  but  it  is  by  no  means  always  easy  to  get  them 
to  display  the  particular  selectivity  that  is  desired.  Those 
that  do  not  display  it  are  of  little  use,  or  of  none  at  all.  I 
have  therefore  suppressed  a  large  number  of  formulae  which 
appear  to  me  to  have  little  or  no  scientific  value.  Most  of 
them,  if  required,  will  be  found  quoted  in  the  previous 
editions. 

281.  Picric  Acid.— I  follow  FLEMMING  (Zeit.  f.  wiss.  Mile.,  i, 
1884,  p.  360)  in  pointing  out  that  picric  acid  is  one  of  the 
most  generally  useful  of  all  secondary  stains.    It  gives  useful 
plasma  stains  with  most  of  the  nuclear  stains,  and  particularly 
with  carmine   and  hsematoxylin.      The  modus  operandi  is  as 
simple  as  possible  :   it  consists  merely  in  adding  picric  acid 
to  the  alcohols  employed  for  dehydrating  the  objects  after 
staining  with  a  nuclear  stain. 


COAL-TAR    PLASMA    STAINS.  213 

It  should  be  borne  in  mind  that  picric  acid  has  consider- 
able power  of  crashing  out  other  anilin  stains ;  and  that  in 
combination  with  hydrochloric  («•<'<!  it  very  greatly  enhances 
the  power  with  which  this  acid  washes  out  carmine  stains. 
It  should,  therefore,  not  be  added  to  the  acidulated  alcohol 
taken  for  differentiating  borax-carmine  stains,  or  the  like, 
but  only  to  the  neutral  alcohol  used  afterwards.  It  has  the 
great  quality,  shared  by  very  few  plasma  stains,  that  it  can 
be  used  for  staining  entire  objects.  And  as  it  is  extremely 
penetrating,  it  is  very  much  indicated  for  the  preparation  of 
such  objects  as  small  Arthropods  or  Nematodes,  mounted 
whole. 

It  can  in  some  cases  be  employed  by  dissolving  it  in  the 
solution  of  another  dye  (see  Picro-carmine,  LEGAL' s  alum- 
carmine,  §  216,  etc.) ;  or  (for  sections)  by  dissolving  it  in  the 
xylol  or  chloroform  used  for  clearing. 

282.  Orange  G. — This  is  the  benzenazo-beta-naphthol- 
disulphonate  of  soda  (to  be  obtained  from  Griibler  & 
Hollborn,  and  not  to  be  confounded  with  about  a  dozen 
other  colours  that  are  on  the  market  under  the  name  of 
t(  Orange/'  with  or  without  a  suffix).  As  indicated  by  its 
chemical  description,  this  is  an  "  acid  "  colour  in  Ehrlich's 
sense ;  and  according  to  FLEMMING  (Arch.  mik.  Anat.,  xxxvii, 
1891,  p.  685)  it  is  also  an  acid  colour  in  the  usual  sense,  its 
solution  in  water  having  an  acid  reaction.  My  sample, 
however,  obtained  from  Griibler  &  Hollborn,  and  stated 
to  be  identical  with  that  supplied  to  Flemming,  shows  no 
sign  of  an  acid  reaction.  MAYER  has  examined  two  samples, 
with  the  same  result.  Probably  Flemming's  sample  was 
not  pure.  The  solutions  do  not  keep  well,  throwing  down 
very  quickly  a  pulverulent  deposit. 

I  think  this  is  one  of  the  most  precise  cytoplasm  stains 
that  I  have  met  with,  but  the  stain  is  a  very  pale  one.  I 
use  a  saturated  solution  in  water,  and  allow  it  to  act  for  five 
or  ten  minutes  (sections  only). 

283.  FLEMMIXG'S  Orange  Method  (Arch.f.  mik.  Anat.,  xxxvii,  1891, 
p.  249 ;  ibid.,  p.  685 ;  Zeit.  f.  wise.  Mik.,  viii,  2,  1891,  p.  223,  and  viii,  3» 
p.  343). — Stain  (for  as  much  as  two  or  three  days,  or  even  weeks  if  you 
want  a  very  powerful  stain)  in  strong  alcoholic  safranin  solution  diluted 
with  anilin  water  (§  272) ;  rinse  in  distilled  water ;  differentiate  in  absolute 


214  CHAPTER    XVI. 

alcohol,  containing  at  most  O'l  per  cent,  of  hydrochloric  acid,  until  hardly 
any  more  colour  comes  away  ;  stain  for  one  to  three  hours  in  gentian  violet 
(§  273) ;  wash  for  a  short  time  in  distilled  water ;  treat  with  concentrated, 
or  at  least  fairly  strong,  aqueous  solution  of  orange  G,  which  "  in  virtue  of 
its  acid  properties  "  washes  out  most  of  the  gentian.  After  at  most  a  few 
minutes,  whilst  pale  violet  clouds  are  still  being  given  off  from  the  sections 
on  agitation,  bring  them  into  absolute  alcohol  until  hardly  any  more  colour 
comes  away,  clear  in  clove  or  bergamot  oil,  and  mount  in  damar  or  balsam 
before  the  last  pale  clouds  of  colour  have  ceased  to  come  away.  The  orange 
must  be  the  orange  G  (last  section). 

This  is  not  a  triple  stain  in  the  sense  of  giving  three  different  colours  in 
the  result ;  it  is  a  nuclear  and  plasmatic  stain  in  mixed  tones ;  the  orange, 
according  to  Plemming,  does  not  act  as  a  separate  stain,  but  as  an  agent  for 
the  differentiation  of  the  gentian  stain.  I  am  unable  to  find  that  it  acts  in 
this  way,  for  I  do  not  find  that  it  washes  out  any  of  the  gentian ;  at  all 
events,  in  my  preparations  no  perceptible  clouds  of  colour  come  away  (but 
see  last  section).  It  seems  to  me  more  probable  that  it  acts  by  forming  a 
"neutral"  colour  with  the  gentian  (see  next  section  and  §  262). 

See  also  FLEMMING  in  Arch.  Anat.  Phys.,  Anat.  Abth.,  1897,  p.  175. 

Never  popular,  this  clumsy  and  uncertain  process  is  now  little  used. 

284.  KEINKE'S  Modification  of  FLEMMING'S  Orange  Method  (Arch, 
/.  mik.  Anat.,  xliv,  2,  1894,  p.  262). — Sections  of  material  fixed  in  liquid  of 

HERMANN  are  put  for  twenty-four  hours  into  a  concentrated  solution  of 
potassium  sulphite.  They  are  washed  with  water  and  stained  for  an  hour 
or  two  in  safranin.  They  are  then  well  washed  with  water,  and  stained  for 
twenty-four  hours  in  a  "  neutral"  mixture  of  gentian  and  orange,  prepared 
as  follows : 

To  a  concentrated  aqueous  solution  of  gentian  violet  are  added  "  a  few 
drops  "  of  a  like  solution  of  orange  G.  The  solution  precipitates  in  part, 
owing  to  the  formation  of  an  imperfectly  soluble  "neutral"  colour;  but 
becomes  almost  clear  again  if  an  excess  of  water  be  added.  A  drop  of  the 
mixture  placed  on  blotting-paper  should  form  a  violet  or  brown  spot  with  a 
narrow  orange  border.  The  solution  is  not  to  be  filtered,  but  the  sections 
are  to  be  stained  in  it  as  it  is  (it  is  impossible  to  make  out  whether  REINKE 
means  the  undiluted  mixture,  or  the  mixture  made  almost  clear  by  addition 
of  water).  It  is  said  that  the  "neutral"  solution  may  be  preserved  for 
future  use  by  adding  to  it  one  third  of  alcohol.  After  staining,  you  differ, 
entiate  rapidly  with  alcohol,  and  clear  with  clove  oil. 

I  have  tried  this  process,  and  obtained  exactly  the  same  results  as  with 
Flemming's  process,  and  so  have  other  workers. 

285.  Metanil  Yellow  (Metanilgelb).— See  GEIESBACH  (Zeit.  f.  wiss. 
Mik.,  iv,  4,  1887,  p.  448;  see  also  Journ.  Roy.  Hie.  Sbc.,  1889,  p.  464).    It 
is  said  to  have  a  certain  affinity  for  various  elements  belonging  to  the  group 
of  the  connective  tissues. 

286.  Sauregelb  (Echtgelb),  Tropseolin  O.,  Crocein,  Gold  Orange 
(see  GRIESBACH,  Arch.f.  mik.  Anat.,  xxii,  p.  132). 


COAL-TAR   PLASMA    STAINS.  215 

287.  Saurefuchsin   (Acid    Fuchsin,   Fuchsin    S,   Acid  Rubin, 
Rubin  S,  Saurerubin,  Acid  Magenta,  Magenta  S). — The  chemical 
description  of  this  acid  colour  has  been  given  (§  262)  :  it  is 
important  not  to  confound  it  with  basic  fuchsin,  as  seems  to 
have  been  done  by  some  writers. 

This  is  one  of  the  best  cytoplasm  stains  that  I  know  of. 
I  use  a  0*5  per  cent,  solution  in  water,  and  allow  it  to  act  on 
sections  for  a  few  minutes.  The  stain  is  sufficiently  resistent 
to  alcohol.  Saurefuchsin  is  also  used  as  a  specific  stain  for 
nerve  tissue  (see  "  Neurological  Methods  "  in  Part  II). 

288.  Saurefuchsin  and  Orange  G. — I  have  had  good  results  by 
mixing  the  aqueous  solutions  of  these  two  dyes,   but  unfor- 
tunately have  not  noted  the  proportions. 

289.  VAN   GIBSON'S  Picro-Saurefuchsin  (from   Zeit.  f.   wiss. 
Mik.j  xiii,  3,  1896,  p.  344). — To  a  saturated  aqueous  solution 
of  Saurefuchsin  is  added  a  few  drops  of  saturated  aqueous 
solution  of  picric  sicicl,  until  the  mixture  has  become  garnet- 
red.      After  staining,  rinse  with  water,  dehydrate,  and  clear 
in  oil  of  origanum.      According   to   MOLLEU  (op.  cit.,  xv,  2, 
1898,  p.  174),  a  good  formula,  due  to  WEIGERT,  is — warm — 
saturated   picric  acid    solution,   150    c.c.  ;    saturated    Saure- 
fuchsin solution,  3  c.c. 

OHLMACBER  (Journ.  Exper.  Hed.,  ii,  1897,  p.  675)  adds  0'5 
per  cent,  of  Saurefuchsin  to  a  saturated  solution  of  picric 
acid  which  has  been  diluted  with  an  equal  quantity  of  water. 
He  uses  this  after  previous  staining  with  gentian  violet. 

290.  The  EHRLICH-BIONDI  Mixture  (or  EHRLICH-BIONDI-HEI- 
DENHAIX  Mixture)    (Pfl tiger's  Arch.,  xlii,   1888,  p.  1  ;   Zeit.  f. 
?'-/.sx.  A/7/»\,  v,    4,    1888,  p.   520). — This   well-known   stain  is 
somewhat  troublesome  to  prepare.      It  may  be  obtained  ready 
made  from  Griibler  &  Hollborn. 

The  receipt  is  as  follows  : — To  100  c.c.  saturated  aqueous 
solution  of  orange  add  with  continual  agitation  20  c.c. 
saturated  aqueous  solution  of  Saurefuchsin  (Acid  Fuchsin) 
and  oO  c.c.  of  a  like  solution  of  methyl  green. 

(According  to  Krause  [Arch.  mik.  Anal.,  xlii,  1893,  p.  59],  100  parts  of 
water  will  dissolve  about  20  of  Saurefuchsin  [Rubin  S],  8  of  orange  G; 
and  8  of  methyl  green.)  The  solutions  must  be  absolutely  saturated, 
which  only  happens  after  several  days. 


216  CHAPTER    XVI. 

Dilute  the  mixture  with  60  to  100  volumes  of  water.  The 
dilute  solution  ought  to  redden  if  acetic  acid  be  added  to 
it ;  and  if  a  drop  be  placed  on  blotting-paper  it  should  form 
a  spot  bluish  green  in  the  centre,,  orange  at  the  periphery. 
If  the  orange  zone  is  surrounded  by  a  broader  red  zone,  the 
mixture  contains  too  much  fuchsin. 

According  to  M.  HEIDENHAIN'S  instructions  ("  Ueber  Kern 
u.  Protoplasma/'  in  Festschr.  Herrn.  Geli.  A.  v.  Kolliker 
gewidm.,  etc.,  1892,  p.  115;  Zeit.  f.  wins.  Hik.,  ix,  2,  1892, 
p.  202)  the  orange  to  be  used  should  be  "  Orange  G ;  "  the 
Acid  Fuchsin  or  Saurefuchsin  should  be  "  Eubin  8 }) 
("  Rubin "  is  a  synonym  of  Fuchsin);  and  the  methyl 
green  should  be  "  Methylgriin  00."  And  it  is  absolutely 
necessary  that  these  ingredients  be  those  prepared  under 
those  names  by  the  Actienfabrik  fur  Aniliu fabrication  in 
Berlin.  They  can  be  obtained  from  Griibler  &  Hollborn, 
either  separately,  or  as  a  mixture  of  the  three  dyes  in 
powder  (which  I  do  not  recommend). 

The  strong  solutions  directed  to  be  taken  readily  precipi- 
tate on  being  mixed.  To  avoid  this  it  is  recommended  by 
SQUIRE  (Methods  and  Formulas,  etc.,  p.  37)  to  dilute  them 
before  mixing. 

Other  proportions  for  the  mixture  have  been  recommended  by  KEAUSE 
(loc.  cit.  supra),  viz.  4  c.c.  of  the  Saurefuchsin  solution,  7  of  the  orange  G, 
and  8  of  the  methyl  green ;  the  mixture  to  be  diluted  50  to  100-fold  with 
water.  THOME  (Arch.  mik.  Anat.,  Hi,  1898,  p.  820)  gives  the  proportions 
2  :  5  :  8,  and  dilutes  100-fold. 

Stain  sections  (N.B.,  sections  only)  for  six  to  twenty-four 
hours.  Dehydrate  with  alcohol,  clear  with  xylol,  and  mount 
in  xylol  balsam. 

In  the  intention  of  the  observers  who  have  elaborated  this 
stain,  it  is  a  progressive  stain,  and  not  a  regressive  one.  It 
does  not  require  any  differentiation,  and  the  sections  should 
be  got  through  the  alcohol  into  xylol  as  quickly  as  possible 
in  order  to  avoid  any  extraction  of  the  colour.  The  great 
point  of  difficulty  in  working  with  this  stain  is  to  prevent 
the  colour  of  the  methyl  green  from  coming  out  in  the 
alcohol. 

The  best  results  are  obtained  with  sublimate  material ; 
chrom-osmium  material,  and  the  like,  give  a  much  inferior 
stain.  Preparations  made  with  the  usual  mixture,,  as  given 


COAL-TAB    PLASMA   STAINS.  2l7 

above,  are  liable  to  fade;  by  acidifying  the  mixture  a 
stronger  and  more  sharply  selective  stain  is  obtained,  which 
does  not  fade.  But  too  much  acid  must  not  be  added,  as 
this  would  cause  a  staining  of  the  iiiterfilar  substances.  The 
following  instructions  for  acidifying,  due  to  M.  HEIDENHAIN, 
are  from  a  paper  of  WARBURG'S  (quoted  from  Zeit.  f.  >'•/**. 
Mil:.,  xi,  3,  1894,  p.  383).  To  2  c.c.  of  the  Biondi  mixture 
(1  :  30)  (by  this  is  presumably  meant  the  original  mixture  as 
given  above,  but  diluted  with  only  30  volumes  of  water 
instead  of  60)  add  40  c.c.  of  distilled  water,  3  c.c.  of  0'5 
per  cent,  solution  of  Saurefuchsin,  and  0'2  c.c.  of  one  fifth 
per  cent,  solution  of  acetic  acid  (or,  according  to  GROOVEN, 
up.  cit.,  xii,  3,,  1896,  p.  379,  four  drops).  [Grouven  here 
speaks  of  the  mixture  as  "  Triacid  "  (see  next  section)  ;  there 
is  a  deplorable  confusion  in  the  nomenclature  of  these 
stains.] 

Another  process  of  acidification,  complicated  and  difficult,  is  given  by 
M.  HEIDENHAIN  ( Veber  Kern  und  Protoplasma,  p.  116 ;  Zeit.f.  iciss.  Mik., 
ix,  2,  1892,  p.  202) ;  for  this  see  last  edition.  See  also  ISRAEL  (Praktilmm 
Path.  Hist.,  2  Aufl.,  Berlin,  1893,  p.  69) ;  TBAMBUSTI  (Ricerche  Lab.  Anat. 
Roma,  v,  1896,  p.  82;  Zeit.  f.  wiss.  Mile.,  xiii,  1896,  p.  357) ;  and  THOME 
(op.  cit.  supra). 

After  acidification  the  solution  must  not  be  filtered,  for 
filtration  may  render  it  less  acid.  If  it  has  been  kept  for 
some  time  a  little  more  acid  must  be  added  ;  for  it  will  have 
dissolved  traces  of  glass,  which  is  an  alkaline  body.  M. 
HEIDENHAIN  therefore  recommends  that  it  be  preserved  in 
rubber  bottles. 

Before  staining  (M.  HEIDENHAIN,  loc.  cit.),  sections  should 
be  treated  for  a  couple  of  hours  with  0*1  per  cent,  acetic 
acid,  then  for  ten  to  fifteen  minutes  with  officinal  tincture  of 
iodine,  and  be  rinsed  with  alcohol  before  bringing  into  the 
stain,  in  which  they  should  remain  for  twelve  to  eighteen 
hours.  The  treatment  with  acid  is  necessary  in  order  to 
ensure  having  the  sections  acid  on  mounting  in  balsam.  The 
primary  object  of  the  iodine  is  to  remove  any  sublimate  from 
the  preparations  (Heidenhain's  descriptions  refer  to  sublimate 
objects),  but  it  also  enhances  the  power  of  staining  of  the 
chromatin  with  methyl  green,  and  produces  a  more  selective 
staining  of  protoplasmic  elements. 


218  CHAPTER   XVI. 

The  stain  is  a  very  fine  one  when  successful.  But  it  is 
very  capricious,  it  seldom  gives  the  same  result  twice  running. 
The  correct  result  should  be  a  precise  chromatin  stain  com- 
bined with  a  precise  stain  of  the  plastin  element  or  reticuluni 
of  cytoplasm  by  the  Saurefuchsin.  Now  the  least  defect  or 
excess  of  acidity  causes  the  plasma  stain  of  the  Saurefuchsin 
to  become  a  diffuse  one,  instead  of  being  sharply  limited  to 
the  plastin  element.  And  the  methyl  green,  being  very 
little  resistent  to  alcohol,  goes  out  of  the  chromatin  Avith 
such  rapidity  during  the  dehydration,  that  there  is  always 
danger  of  the  chromatin  stain  being  lost  altogether.  For 
this  reason  the  stain  will  only  work  with  very  thin  sections  : 
to  be  quite  sure  of  good  results,  the  sections  should  be  of 
not  more  than  3  /n  in  thickness,  and  if  they  are  over  five  the 
desired  results  are  almost  hopeless.  The  preparations  keep 
very  badly  ;  the  majority  of  mine,  at  any  rate,  have  become 
spoilt  sooner  or  later,  sometimes  after  only  a  few  days.  I 
admit  that  the  method  has  its  raison  d'etre  for  the  very 
special  objects  for  which  it  was  imagined, — for  the  researches 
on  cell-granulations  for  which  EHRLICH  employed  the  three 
colours,  or  for  the  researches  on  the  plastin  reticuluni  of 
cytoplasm  for  which  MARTIN  HEIDENHAIN  employed  the 
mixture ;  for  the  study  of  gland  cells ;  and  for  similar 
objects.  But  to  recommend  it  and  to  use  it,  as  has  been 
done  by  many  workers,  as  a  general  stain  for  sections, 
applicable  to  ordinary  work,  is  nothing  but  mischievous 
exaggeration.  Far  from  having  the  qualities  that  should  be 
possessed  by  a  normal  section-stain,  the  Ehrlich-Biondi 
mixture  is  highly  unfitted  for  ordinary  work.  Workers 
have  at  length  found  this  out,  and,  after  a  period  of  well- 
nigh  unparalleled  popularity,  this  stain  is  now  but  little  used 
except  for  the  special  purposes  above  indicated. 

291.  EHRLICH'S  Triacid  Mixture. — According  to  a  custom 
whicji,  I  believe,  originated  with  Ehrlich  himself,  and  which 
would,  perhaps,  be  "  better  honoured  in  the  breach  than  the 
observance/'  the  name  of  Triacid  ("  Triacidlosung "}  has 
been  given  to  a  mixture  of  the  same  three  dyes  as  in  the 
EHRLICH-BIONDI  mixture,  but  in  such  proportions  that  the 
"  acid  "  colours  therein  have  a  larger  share  assigned  to  them. 
The  denomination  is  improper,  for  the  mixture  contains  only 


COAL-TAE   PLASMA  STAINS. 

two  "  acid  "  colours,  methyl  green  being  a  strongly  "  basic  " 
colour.  The  following  is  a  recent  formula  of  EHRLICH,  com- 
municated to  REINBACH  (quoted  from  Zeit.  f.  wiss.  Mik.,  xir 
3,  1894,  p.  259). 

Orange  G,  sol.  sat.  aq.       .  .  .120 

Saurefuchsin         „  ...        80 

Methyl  green       „  ...      100 

Distilled  water          ....      300 
Absolute  alcohol        .  .  .  .180 

Glycerin  .....        50 

Care  must  be  taken  that  the  solutions  be  absolutely  satu- 
rated before  mixing.  The  mixture  must  never  be  shaken, 
the  quantity  necessary  for  use  at  any  time  must  be  carefully 
taken  off  from  the  top  of  the  stock  by  means  of  a  pipette. 
With  these  precautions  the  mixture  will  keep  for  years. 

I  have  not  made  up  this  mixture,  but  have  examined  a 
triacid  solution  procured  from  Griibler  &  Hollborn.  Its 
qualities  and  defects  appear  to  be  very  much  those  of  the 
Ehrlich-Biondi  mixture.  It  appears  to  be  a  more  powerful, 
but  perhaps  less  delicate  stain,  and  the  methyl  green  appears 
to  have  more  resistance  to  alcohol,  so  that  it  may  perhaps 
give  better  results  for  ordinary  work. 

292.  Bordeaux  E. — A  general  stain  taking  effect  both  on 
chromatin  and  cytoplasm,  and,  I  consider,  a  very  good  plasma 
stain.  It  is  much  used,  011  the  recommendation  of  M. 
HEIDENHAJN  (Arch.  mih.  Atiat.,  xlii,  1894,  p.  665),  for  stain- 
ing sections  before  iron-haematoxylin,  it  being  supposed  that 
this  treatment  affords  a  sharper  stain  of  his  "  central  cor- 
puscles/7 I  use  for  chrom-osmium  material  a  1  per  cent, 
solution,  and  stain  for  twelve  to  twenty-four  hours.  The 
stain  resists  alcohol  well. 

293.  Bordeaux  R,  Thionin,  and  Methyl  Green  (GEABEBG,  Zeit.f. 
wise.  Mik.,  xiii,  4,  1896,  p.  460). 

294.  Congo  Red  (Congoroth)  (see  GBIESBACH,  in  Zeit.f.  wiss.  Mik.,  iii, 
3, 1866,  p.  379).— Also  an*"  acid  "  colour  in  EHBLTCH'S  sense.     The  aqueous 
solution/however,  Has  a  neutral  or  alkaline  reaction.     It  becomes  blue  in 
presence  of  the  least  trace  of  free  acid  (hence  Congo  is  a  valuable  reagent 
for  demonstrating  the  presence  of  free  acid  in  tissues;  see  the  papers  quoted, 
loc.  cit.}.     A  stain  much  of  the  same  nature  as  Saurefucbsin,  and  like 


220  CHAPTER    XVI. 

it  useful  in  staining  axis-cylinders.  See  the  chapter  on  Nerve  Tissue,  in 
Part  II.  It  may  also  be  used  for  staining  some  objects  during  life  (see 
ante,  §  201).  1  have  tried  it  as  a  general  plasma  stain,  and  cannot  recom- 
mend it,  as  the  stain  does  not  keep.  CAENOY  (La  Cellule,  xii,  2,  1897, 
p.  216)  has,  however,  had  very  good  results  with  it  as  a  secondary  stain 
employed  after  hamatoxylin  of  DELAFIELD,  and  found  the  stain  keep 
well.  He  used  a  O'o  per  cent,  solution  in  water.  Note  that  this  colour 
is  not  to  be  confounded  with  other  Congos,  as  Congo  yellow,  or  Brilliant 
Congo. 

LOISEL  (Journ.  de  I'Anat.  et  de  la  Phys.,  1898,  p.  230)  says  concerning 
the  reaction  with  free  acids,  that  the  colour  is  azure-blue  with  mineral 
acids  and  dark  violet  with  organic  acids.  In  the  presence  of  chlorine 
compounds  it  gives  the  same  reaction  as  with  acids.  In  the  presence  of 
ammoniacal  liquids  it  will  not  give  the  reaction  with  C02  nor  with  acetic  or 
lactic  acid.  See  WUESTEE,  Centralb.f.  PhysioL,  1887,  p.  240. 

295.  Benzopurpurin. — According  to  GEIESBACH  (loc.  cit.),  another 
"acid"  colour  very  similar  in  its  results  to  Congo  red.  It  has  been  commended 
as  a  plasma  stain.  I  have  myself  been  unable  to  obtain  any  results  what- 
ever with  it.  See,  however,  ZSCHOKKE  (ibid.,  v,  4, 1888,  p.  466),  who  recom- 
mends Benzopurpurin  B,  and  says  that  weak  aqueous  solutions  should 
be  used  for  staining,  which  is  effected  in  a  few  minutes,  and  alcohol  for 
washing  out.  Deltapurpurin  has,  it  is  said,  similar  properties,  and  may 
be  used  in  the  same  way. 

296.  Neutral  Red  (Neutralroth)  (EHRLICH,  Allg.  med.  Zeit., 
1894, pp.  2,  20;  Ze-it.f.  >iciss.  Milc.,xi,  2,  1894,  p.  250;  GALEOTTI, 
ibid.,  p.  193). — Up  to  the  present  this  colour  has  chiefly 
been  employed  for  intra-vitam  staining.  Tadpoles  kept  for  a 
day  or  two  in  a  solution  of  1  :  10,000  or  100,000  absorb  so  con- 
siderable a  quantity  of  the  colour  that  all  their  tissues  appear 
of  a  dark  red.  The  stain  is  limited  to  cytoplasmic  granules 
(EHRLICH),  and  to  the  contents  of  mucus  cells  (GALKOTTI). 

S.  MAYER  (Lotos,  Prague,  1806,  No.  2)  states  that  it  also 
stains  degenerating  myelin.  According  to  a  further  study 
of  this  colour  by  EHRLICH  and  LAZARUS  (Spec.  Pathol.  und 
Therapie,  herausgeg.  von  NOTHNAGEL,  viii,  1,  1898,  p.  1  ; 
Zeit.  f.  wixs.  Mile.,  xv,  3,  1899,  p.  338)  it  may  be  used  for 
intra-vitam  staining  of  tissues  in  the  same  way  us  methylen 
blue  (next  chapter),  by  injection  or  immersion  with  contact 
of  air.  It  is  especially  a  granule  stain.  Similar  results  are 
recorded  by  ARNOLD  (Anat.  Anz.,  xvi,  1899,  p.  568).  See 
also  LOISEL  (Journ.  de  VAnat.  et  de  la  PhysioL,  1898, 
pp.  197,  210,  217)  (intra-vitam  staining  of  sponges)  ;  and 
PROWAZEK,  Zeit.  f.  wiss.  ZooL,  Ixii,  1897,  p.  187  (intra-vitam 


COAL-TAR  PLASMA    STAINS.  221 

-raining  of  Protozoa).      I  myself  have  had  very  good  results 
with  it  as  an  intra-vitam  stain. 

It  has  ajso  been  found  useful  for  staining,  in  hardened 
material,  the  corpuscles  of  NISSL  (q.  v.)  in  nerve-cells. 
These  bodies  are  basophilous;  hence,  it  is  suggested,  their 
staining  by  neutral  red,  which  is  a  "  basic  "  colour.  Tha 
term  "  neutral "  refers  to  the  hue  of  its  solution,  not  to 
its  chemical  composition.  Its  neutral  red  tint  is  turned 
bright  red  by  acids,  yellow  by  alkalies.  The  stain  in  tissues 
is  in  general  metachromatic,  nuclei  being  red,  cell-bodies 
yellow  (cf.  ROSIN,  in  Deutsche  med.  Wochenschr.,  xxiv,  1898, 
p.  615  ;  Zeit.  f.  wiss.  Mik.,  xvi,  2,  1899,  238).  The  solutions 
that  have  been  employed  for  staining  fixed  material  are 
strong  aqueous  ones,  1  per  cent,  to  concentrated.  See  further 
under  NISSL,  in  the  chapter  on  " Neurological  Methods." 

297.  Biebricher  Scharlach  (BIEBEICH  SCAELET),  a  diffuse  bright  red 
stain,  may  possibly  be  useful  as  a  contrast  stain.  See  GBIESBACH,  Arch.  f. 
mik.  Anat.,  xxii,  p.  132. 

298.  The  Eosins,  found  in  commerce  under  the  names  of 
Eosin,  Saffrosin,  Primerose  Soluble,  Phloxin,  Bengal  Rose,  Ery- 
throsin,  Pyrosin  B,  Rose  B,  a  1'Eau,  etc.,  are  all  phthalein 
colours.  The  preparations  indicated  by  these  names  are  not 
quite  identical  in  their  properties,  but  vary  according  to  the 
different  modes  of  manufacture.  Most  of  them  are  soluble 
both  in  alcohol  and  in  water,  but  some  only  in  alcohol  ("  Pri- 
merose a  I' Alcohol"). 

They  are  all  diffuse  stains,  formerly  much  used  as  con- 
trast stains,  less  so  now. 

Their  chief  use  isJn  combinations  or  mixtures,  to  be  given 
further  on.  For  Bengal  Rose  see  GTRIESBACH,  Zool.  Anz., 
No.  135,  1883,  p.  172. 

Eosin  is  a  specific  stain  for  red  blood-corpuscles,  and  also 
for  certain  granules  of  leucocytes,  and  hence  is  much  used 
in  the  study  of  blood,  for  which  see  in  Part  II. 

The  yolk  of  some  ova  takes  the  stain  strongly,  so  that  it 
is  useful  in  some  embryological  researches. 

299.  Methyl  Green  and  Eosin  (CALBEBLA,  Morph.  Jahrb.,  iii,  1877, 
Heft  3,  p.  625;  LIST,  Zeit.f.  wiss.  Mik.,  ii,  1885,  p.  147;  BALBIANI,  Ann. 
Microgr.,  Paris,  vii,  1895,  p.  245 ;  RHUMBLEE,  Zeit.  wiss.  Zool,  Ixi,  1895, 
p.  38). — See  previous  editions. 


222  CHAPTER   XVI. 

300.  Methylen  Blue  and  Eosin  (CHENZINSKY,  quoted  from  Zeit.f. 
wiss.  Mik.,  xi,  2,  1894,  p.  260). 

Methylen  blue,  sol.  sat.  in  water  .  .  .  .40 
Eosin,  0'5  per  cent,  in  70  per  cent,  alcohol  .  .  20 
Distilled  water,  or  glycerin 40 

This  solution  will  only  keep  for  about  eight  days. 

It  has  been  recommended  as  a  specific  stain  for  blood. 

The  mixture  of  PIANESE  (Zeit.f.  wiss.  Mik.,  xi,  3,  1894,  p.  345)  contains 
the  same  ingredients  in  the  same  proportions,  with  the  addition  of  a  con- 
siderable proportion  of  carbonate  of  lithia. 

See  also  the  mixture  of  BEEMER,  Arch.f.  mik.  Anat.,  xlv,  1895,  p.  433  ; 
or  Zeit.f.  wiss.  Mik.,  xii,  3,  1896,  p.  380. 

I  have  tried  CHENZINSKY'S  mixture  as  a  tissue  stain,  without  good  re- 
sults; but  see  ROSIN,  Berliner  Min.  Wochenschr.,  1898,  p.  251;  Zeit.f. 
wiss.  Mik.,  xvi,  2,  1899,  p.  223. 

301.  Light  Green  (Litchgriin  S.  F.). — An  acid  colour,  in 
Ehr-licn's  sense,  soluble  in  water  or  alcohol,  and  a  good 
plasma  stain. 

BKNDA  (Verh.  physiol.  Ges.  zu  Berlin,  Dec.  18th,,  1891, 
Nos.  4  u.  5  ;  see  also  Zeit.  f.  wiss.  Mik.,  viii,  1892,  p.  516) 
stains  sections  for  twenty-four  hours  in  aniliii-water  safranin 
solution,  then  for  about  half  a  minute  in  a  solution  of  0'5 
grm.  Lichtgriin  or  Saureviolett  (Griibler)  in  200  c.c.  of 
alcohol,  dehydrates  and  mounts  in  balsam.  This  process 
gives  one  of  the  most  beautiful  stains  known  to  me.  The 
Saureviolett  gives,  perhaps,  the  more  brilliant  preparations, 
and  seems  to  be  rather  easier  to  carry  out,  as  it  may  be 
allowed  to  act  rather  longer  than  the  Lichtgriin.  The  pro- 
cess in  either  form  is  a  rather  delicate  one  to  carry  out,  and 
requires  very  thin  sections.  The  Lichtgriin  stain  unfor- 
tunately does  not  keep  very  well ;  I  find  my  preparations 
much  faded  after  two  years,  but  they  will  keep  sufficiently 
for  many  months  at  all  events. 

302.  Malachite  Green  (syn.  Solid  Green,  Victoria  Green,  New 
Green,  Benzoyl  Green,  Fast  Green). — A  basic  colour,  which  has  been 
used  as  a  plasma  stain  for  the  ova  of  Ascaris  by  VAN  BENEDEX  and  NEYT 
(see  in  Part  II,  "  Cytological  Methods  ").    These  authors  used  it  for  glycerin 
preparations.     I  have  tried  it  for  balsam  sections,  and  find  that  it  cannot  be 
used,  as  the  stain  is  not  sufficiently  resistent  to  alcohol. 

303.  Iodine  Green  ("  HOFMANN'S  Grim"),  see  GBIESBACH  (Zool.  Anz., 
No.  117,  vol.  v,  1882,  p.  406).— The  colour  is  now  no  longer  manufactured 

ial  purposes,  but  it  is  said  may  be  obtained  of  excellent  quality 


COAL-TAR    PLASMA    STAINS.  223 

from  C.  A.  F.  Kahlbauin's  Chemische  Fabrik,  Berlin,  S.C.  (Zool.  Anz.,  No. 
130,  1883,  p.  56).  Stain  essentially  that  of  methyl  green,  but  plasma  often 
violet  through  the  presence  of  a  violet  impurity  (MATES,  Mitth.  Zool.  Stat. 
Neapel,  xii,  1896,  p.  311 ;  see  also  earlier  editions). 

304.  Thiophen  Green  (Thiophengrun),  see  KBAUSE,  Intern.  Monats- 
schr.f.  Anat.,  etc.,  iv,  1887,  Heft  2. 

305.  Anilin  Green  is  said  to  have  a  special  affinity  for  mucous  gland 
cells,  and  other  qualities ;  but  I  have  not  been  able  to  identify  the  colour 
mentioned  by  authors. 

306.  Coerulein  S.,  a  green  dye,  is  recommended  for  the  staining  of 
muscle-fibrils  by  M.  v.  LENHOSSEK  (Anat.  Anz.,  xvi,  1899,  p.  339). 

307.  Quinolem  Blue  (Cyanin,  Chinolinblau ;  v.  RAXVIER,  Traite, 
p.  102).— Quinolein  is  said  to  have  the  property  of  staining  fatty  matters 
an  intense  blue. 

It  is  useful  for  staining  Infusoria,  which  in  dilute  solution  it  stains 
during  life.  On  this  point  see  the  methods  of  CEETES  (post,  Part  II). 

308.  Indulin  and  Wigrosin. — The  Indulins  are  a  group  of  dyes  related 
to  the  base   violanilin.      They  may  be  either  •'  basic "  colours  or  "  acid  " 
colours.     The  soluble  indulins  of  commerce  are  generally  mono-  and  disul- 
phonic  acids   (BENEDIKT   and   KXECHT,    Chemistry  of  Coal-tar  Colours, 
p.  187).     They  occur  under  the  brands  (not  strictly  synonyms)  of  Indulin, 
Nigrosin,  Indigen,   Coupler's  Blue,  Fast  Blue   R,  Fast  Blue  B, 
Blackley  Blue,  Guernsey  Blue,  Indigo  substitute.     According  to 
BEHBEXS  the  name  Indulin  is  generally  given  to  a  bluish  brand,  and  that 
of  Nigrosin  to  a  blacker  one. 

Nigrosin,  used  with  sublimate  material,  I  find  stains  both  nuclei  and 
cytoplasm,  the  chromatin  strongly.  I  do  not  consider  it  a  very  good  plasma 
stain.  It  will  not  give  the  stain  at  all  with  chrorn -osmium  material. 

According  to  CALBEELA  (Morpli.  Jahrb.,  iii,  1877,  p.  627)  the  concentrated 
aqueous  solution  of  Indulin  should  be  diluted  with  six  volumes  of  water. 
Sections  will  stain  in  the  dilute  solution  in  five  to  twenty  minutes;  they 
may  be  washed  in  water  or  in  alcohol,  and  examined  either  in  glycerin  or 
oil  of  cloves. 

The  peculiarity  of  this  stain  is  said  by  CALBEELA  to  be  that  it  never  stains 
nuclei ;  the  remaining  cell-contents  and  intercellular  substance  are  stained 
blue.  In  its  general  effects  it  resembles  quinolein  blue,  and  is  exactly  the 
opposite  of  methyl  green.  As  far  as  I  have  been  able  to  control  CALBEBLA'S 
assertions,  they  appear  to  me  correct ;  but  I  see  no  special  good  in  it. 

309.  EHRLICH'S  Indulin-Aurantia-Eosin,  or  Acidophilous  Mix- 
ture, or  Mixture  C,  or  Mixture  for  Eosinophilous  Cells  (from  the 
formula  kindly  sent  me  by  Dr.  GRUBLER). — Indulin,  auraii'iiu, 


224  CHAPTER    XVI. 

and  eosin,  of  each  two  parts ;  glycerin,  thirty  parts.  This 
gives  a  very  thick,  syrupy  solution.  To  use  it,  cover- glass 
preparations  may  be  floated  on  to  it ;  or  sections  on  slides 
may  have  a  few  drops  poured  on  to  them,  the  slide  being* 
laid  flat  till  the  stain  has  taken  effect  (twenty-four  hours 
for  Flemming  material).  This  mixture  was  imagined  for 
the  purpose  of  obtaining  a  specific  stain  of  certain  granules 
of  leucocytes.  It  has  been  pointed  out  by  NIKIFOEOW  (Zeit. 
f.  wiss.  Mik.,  viii,  2,  1891,  p.  189  ;  and  xi,  2,  1894,  p.  246) 
that  it  is  also  available  for  staining  sections.  I  find  this  is 
the  case.  With  Flemming  material  it  gives  a  powerful  and 
good  stain,  which  is  much  more  resistent  to  alcohol  than  that 
of  the  EniiLiCH-BioNDi  mixture,  and  is  therefore  much  more 
adapted  to  ordinary  work.  Chromatin  in  my  preparations 
is  of  a  very  dark  blue,  cytoplasm  being  of  a  lighter  blue 
(except  where  it  has  taken  the  stain  of  the  aurantia  or 
eosin).  It  will  thus  be  seen  that  the  Indulin  in  this  com- 
bination behaves  in  a  manner  quite  opposed  to  its  behaviour 
when  used  alone  (see  last  §).  The  stain  is  said  to  keep 
well. 

ISRAEL  (Praktik.  Path.  Hist.,  Berlin,  1893,  p.  68)  gives  a  more  compli- 
cated receipt. 

310.  Safranin  and  Indigo- Carmine  or  Nigrosin  (KossiNSKi,  Zeit. 
f.  wiss.  Mik.,  vi,  1,  1880,  p.  61). — See  previous  editions. 

311.  Anilin  Blue. — Under  this  title  are  comprised  various 
derivatives  of  the  base  rosanilin.  They  occur  under  the 
names  Spirit  Soluble  Blue  (Bleu  Alcool),  Gentian  Blue  6  B,  Spirit 
Blue  0,  Opal  Blue,  Bleu  de  Nuit,  Bleu  Lumiere,  Parma  Blue,  Bleu 
de  Lyon.  Receipts  of  the  older  authors  for  staining  with 
"  Anilin  blue  "  should,  I  think,  be  disregarded,  as  it  would 
probably  now  be  impossible  to  obtain  the  colours  used  by 
them,  or  even  to  ascertain  what  colour  was  meant  by  them. 

The  only  one  of  the  above-mentioned  colours  of  which  I 
have  any  personal  knowledge,  or  that  appears  really  valuable, 
is  Bleu  de  Lyon.  (Some  authors  give  the  names  Bleu  de  Nuit 
and  Griinstichblau  as  synonyms  of  Bleu  de  Lyon.)  I  find 
that  though  there  is  nothing  very  specific  in  its  action,  it  is 
a  very  good  plasma  stain.  It  is  a  fairly  true  plasma  stain, 
for  though  in  a  strong  stain  it  will  stain  chromatin,  yet  in  a 


COAL-TAR    PLASMA   STAINS.  225 

light  stain  it  will  stain  cytoplasm  first,  and  thus  works  very 
well  with  carmine  or  safranin  as  a  nuclear  stain,  leaving  the 
chromatin  of  a  perfectly  pure  red.  I  find  it  gives  very  good 
differentiations  of  nerve  tissue,  and  of  cartilage  (as  has 
already  been  pointed  out  by  BAUMGAETEN  and  by  JACOBY). 
The  older  mode  of  using  it  (MAUEICE  and  SCHULGIN)  was  to 
stain  in  bulk  with  it  after  borax-carmine,  using  a  very  dilute 
alcoholic  solution.  BAUMGARTKN  and  JACOBY  stain  sections  in 
a  0'2  per  cent,  alcoholic  solution.  I  have  experimented 
with  safranin  as  the  chromatin  stain,  and  obtained  some  fair 
results.  But  I  do  not  think  the  combination  can  be  recom- 
mended. For  if  you  stain  first  with  the  blue,  the  safranin 
will  extract  it  if  allowed  to  act  for  more  than  a  short  time. 
And  if  you  stain  first  with  the  safranin,  the  blue  will  extract 
it  very  quickly. 

312.  Methyl  Blue. — Under  this  title  are  comprised  some 
other  derivatives  of  the  base  rosanilin.  They  are  "  acid  " 
colours,  being  mostly  salts  of  triphenylrosanilin-  and  tri- 
pheiiyl-pararosanilin-trisulphonic  acid.  Here  belong  the  dyes 
sold  as  Methyl  Blue,  Cotton  Blue,  Water  Blue  (Wasserblau), 
Methyl  Water-Blue,  China  Blue  (Chinablau),  Soluble  Blue. 

Amongst  these  Water  Blue  (Wasserblau)  possesses  some 
useful  properties.  According  to  MITKOPHANOW  (quoted  from 
Zeit.  f.  wiss  Mik.,  v,  4,  1888,  p.  513),  used  in  concentrated 
aqueous  solution  it  gives  a  very  good  double  stain  with 
safranin.  It  is  very  resistent  to  alcohol.  Using  the  Wasser- 
blau first,  and  then  the  safranin,  I  have  had  some  interesting 
results,  and  as  the  process  is  easy  to  carry  out  I  think  it  may 
be  recommended.  The  Wasserblau  must  be  used  first,  as  if 
used  after  the  safranin  it  will  destroy  the  stain  in  a  short 
time.  With  chrom-osmium  material,  twelve  to  twenty-four 
hours  in  the  blue,  and  four  or  five  in  the  safranin,  may  not 
be  too  much.  My  stains  have  not  kept  well. 

MANN  (op.  cit.,  xi,  4,  1894,  p.  490)  has  used  it  in  conjunction  with  eosin 
for  staining  ganglion  cells.  For  the  somewhat  complicated  details  of  the 
process,  see  the  place  quoted. 

313.  Anilin  Blue-black  — A  preparation  cited  under  this  name  has 
been  recommended  by  BEVAN  LEWIS  and  others  for  nervous  tissue.  Unfor- 
tunately these  authors  have  not  given  the  chemical  description  of  the 

15 


226  CHAPTER   XVI. 

colour  used  by  them,  so  that  it  is  not  possible  to  ascertain  whether  they 
mean  the  Blue-black  B  of  the  oxyazo  series,  or  the  Anilin  black  of 
LIGHTFOOT,  also  known  under  the  names  of  Nigranilin  and  Noir  Colin. 
Dr.  GEUBLEE  writes  me  that  the  anilin  blue-black  of  his  list  is  the  oxyazo 
colour  bine-black  B  or  AZOSCHWAEZ,  and  that  he  believes  that  Nigranilin 
and  Noir  Colin  are  no  longer  manufactured. 

314.  Carmine   Blue  (Bleu  Carmiii  Aqueux,  prepared    by  Meister, 
Lucius  and  Briinig,  at  H6chst-a-M.). — JANSSENS  (La  Cellule,  ix,  1,  1893, 
p.  9)  has  shown  that  this  colour  possesses  a  special  affinity  for  the  parts  of 
cytoplasm  that  are   undergoing  cuticular  differentiation.      He  uses  it  in 
alcoholic  solution  acidified.     The  stain  will  bear  mounting  in  balsam. 

315.  Violet  B  (or  Methyl  violet  B)  (S.  MAYEE,  Sitzb.  d.  le.  fc.  Akad. 
d.  wiss.  Wien,  iii  Abth.,  February,  1882). — This  colour  is  a  methyl  violet 
prepared  by  Bindschedler  and  Busch  of  Bale,  and  by  the  Aktienfabrik  fur 
Anilinfarben  at  Berlin.     Used  in  solutions  of  1  gnu.  of  the  colour  to  300 
grms.  of  0'5  per  cent,  salt  solution,  and  with  fresh  tissues  that  have  not 
been  treated   with  any  reagent   whatever,  this   colour   gives   a   stain    so 
selective  of  the  elements  of  the  vascular  system  that  favourable  objects, 
such  as  serous  membranes,  appear  as  if  injected.     The  preparations  do  not 
keep   well  ;    acetate   of   potash    is   the   least   unsatisfactory   medium   for 
mounting  them  in,  or  a  mixture  of  equal  parts  of  glycerin  and  saturated 
solution  of  picrate  of  ammonia  (Anat.  Anz.,  1892,  p.  221). 

316.  Saureviolett,  see  §  301. 

317.  Benzoazurin  may  be  made  to  give  either  a  diffuse  or  a  nucleai 
stain,  according  to  MABTIN  (see  Journ.  Roy.  Mic.  Soc.,  1890,  p.  114). 

318.  Baumgarten's   Fuchsin   and  Metliylen  Blue  (Zeit.  f.   wiss. 
Mik.,  i,  1884,  p.  415). — Stain  sections  (of  chromic  objects)  for  twenty-four 
hours  in  a  stain  made  by  adding  8  to  10  drops  of  concentrated  alcoholic 
solution  of  fuchsin  to  a  watch-glassful  of  water.     Einse  with  alcohol,  and 
stain  for  four  or  five  minutes  in  concentrated  aqueous  solution  of  methy- 
len  blue,  wash  out  with  alcohol  for  five  to  ten  minutes,  and  clear  with 
clove  oil.     Nuclei  red,  tissues  blue,  by  substitution. 

319.  RAWITZ'  "Inversion"  Plasma  Stains.— It  has  been  discovered 
by  RAWITZ  that  by  means  of  appropriate  mordants  certain  basic  anilins, 
which  by  the  usual  methods  of   regressive  staining  are   pure   chromatin 
«tams,  may  be  made  to  afford  a  pure  plasma   stain— one    not   affecting 
chromatin  at  all,  thus  giving  an  «  inversion  "  of   the  usual  stain.      The 
stain,  in  my  opinion,  is  a  vile  one.     For  details  of  the  process  see  last 
edition,  or  RAWITZ  (Sitzb.  Ges.  naturf.  Freunde,  Berlin,  1894    p    174- 
Zeit.  f.  wiss.  Mik.,  xi,  4  1895,  p.  503 ;  and  his  Leilfaden  f.  hist.   Unter- 
suchungen,  Jena,  1895,  p.  76). 


COAL-TAR    PLASMA    STAINS.  227 

320.  Artificial  Alizarin  (RAWITZ,  Anat .  Anz.,  xi,  10, 1895,  p.  294).— 
RAWITZ  has  also  worked  out  a  process  of  obtaining  a  double  stain 
(chromatin  and  cytoplasm  being  stained  of  different  colours)  by  means  of 
artificial  Alizarin,  or  Alizarin -cyan  in.  The  process  is  an  adjective  one, 
requiring  the  use  of  special  mordants  supplied  by  the  colour  manufacturers 
and  is  as  follows  : 

Both  the  Alizarin  and  the  mordants  are  those  prepared  by  the  "  Farb- 
werke  vorm.  Meister  Lucius  &  Briining  in  Hochst,"  and  may  be  pro- 
cured through  Griibler  &  Hollborn.  The  Alizarin  should  be  Alizarin 
1,  or  SDG,  or  RX.  A  5  per  cent,  suspension  of  it  (it  will  not  dissolve) 
should  be  made  in  distilled  water.  The  mordant  should  be  that  known  as 
CHROMBEIZE  G  A  I  or  GA  III.  A  stock  solution  should  be  made  by  mixing 
70  parts  of  it  with  130  of  distilled  water. 

Sections  of  Flemming  material  are  brought  into  some  of  the  stock 
solution  of  mordant  diluted  with  an  equal  volume  of  water ;  sections  from 
chromic  or  chrome-picro-nitric  material  into  the  same  diluted  with  2  to  4 
vols.  of  water ;  sections  from  piero-nitric  material  into  the  same  diluted 
with  6  to  10  vols.  of  water.  In  either  case  they  remain  in  the  mordant  for 
twenty-four  hours,  and  are  then  washed  in  distilled  water  until  no  more 
colour  comes  away  from  them. 

They  are  then  brought  into  the  staining  bath.  This  consists  for 
Flemming  material  of  a  portion  of  the  stock  suspension  of  Alizarin  diluted 
with  1  vol.  of  water  ;  for  chromic  and  chrome-picro-nitric  material  of  the 
same  diluted  with  2  to  4  vols.  of  water;  for  picro-nitric  material  of  the 
same  diluted  with  6  to  10  vols.  of  water.  To  each  staining  bath  there  are 
added  a  few  drops  of  1  per  cent,  solution  of  acetate  of  calcium  (to  be 
procured  from  the  Chemische  Fabrik  A.  F.  Kahlbaum,  35,  Schlesischstrasse, 
Berlin,  S.  0.).  They  remain  in  the  bath  for  twenty-four  hours  in  the 
warm,  i.e.  at  a  temperature  of  about  40°  C.  They  are  then  washed  for  half 
an  hour  to  an  hour  in  distilled  water,  then  for  one  to  two  hours  in  96  j>er 
cent,  alcohol,  and  are  cleared  with  bergamot  oil  and  mounted  in  balsam. 
The  sections  must  remain  in  the  alcohol  until  all  excess  of  Alizarin  has  been 
removed  from  them,  which  may  be  known  by  their  becoming  clear  and  no 
longer  covered  with  a  sort  of  fog. 

In  the  result,  chromosomes  ought  to  be  deep  reddish  brown,  plasma 
orange. 

RAWITZ  also  gives,  loc.  cii.,  another  process,  with  "  Alizarin-cyanin  RRR 
doppelt,"  procured  from  the  "  Farbenfabriken  vorm.  Friedrich  Bayer  & 
Co.  in  Elberfeld."  Sections  are  mordanted  for  twenty-four  hours  in  liquor 
ferri  sulfurici  oxydati  (see  §  254)  diluted  with  5  to  20  vols.  of  distilled 
water,  and  are  well  washed  with  distilled  water  before  coming  into  the 
staining  bath.  This  consists  of  a  5  per  cent,  suspension  of  the  dye,  diluted 
with  water  just  as  for  Alizarin,  and  with  acetate  of  calcium  added  to  it  in 
the  same  way.  They  are  stained  therein  and  further  treated  in  all  respects 
as  directed  for  Alizarin.  The  stain  is  a  blue  one. 


CHAPTER  XVII. 

METHYLEN   BLUE. 

321.  Methylen  Blue  is  the  chloride  or  the  zinc  chloride 
double  salt  of  tetramethylthionin,  and  is  a  "  basic  "  colour  in 
EHRLICH'S  sense.  It  appears  that  some  persons  have  con- 
founded it  with  methyl  blue,  to  which  it  has  not,  histologi- 
cally,  any  resembance. 

Commercial  methylen  blue  sometimes  contains  as  an  im- 
purity a  small  quantity  of  a  red  dye,  which  used  to  be  taken  to 
be  methylen  red.  This  impurity  is  present  from  the  beginning 
in  many  brands  of  methylen  blue,  is  frequently  developed 
in  solutions  of  the  dye  that  have  been  long  kept  (so-called 
"  ripened  "  solutions),  and  is  still  more  frequently  found  in 
kept  alkaline  solutions.  It  is  a  reddish-violet  dye,  and  ac- 
cording to  NOCHT  (Centralb.  f.  Bacterial.,  xxv,  1899,  pp. 
764—769  ;  Zeit.  f.  wiss.  Mik.,  xvi,  2,  1899,  p.  225)  it  is  not 
methylen  red,  nor  methylen  violet  either,  but  a  new  colour, 
for  which  NOCHT  proposes  the  name  "  Roth  aus  Methylen- 
blau."  The  presence  of  this  impurity  in  methylen  blue  is 
not  always  an  undesirable  factor ;  .on  the  contrary,  it  some- 
times affords  differentiations  of  elements  of  tissues  or  of  cells 
that  cannot  be  produced  by  any  other  means.  Methylen 
blue  that  contains  it  is  known  as  polychromatic  methylen  blue, 
and  is  employed  for  staining  certain  cell-granules.  It  can 
be  obtained  from  Griibler  &  Hollborn.  (See  also  UNNA'S 
method  of  producing  it,  in  Part  II.) 

The  colour  to  be  employed  for  intra-vitam  nerve  staining 
should,  on  the  contrary,  be  as  pure  as  possible.  APATHY  has 
a  note  in  Zeit.  f.  wiss.  Mik.,  ix,  4,  1893,  p.  466,  to  the  effect 
that  the  best  methylen  blue  for  impregnation — in  fact,  the 
only  one  that  will  give  exactly  the  results  described  by  him 
(see  §  327) — is  that  obtained  from  E.  MERCK,  of  Darmstadt, 


MKTHYLEN    BLUE.  229 

and  quoted  in  his  price  list  as  "  medicinisches  Methylenblau," 
and  described  on  the  label  as  "  Anilin-blau,  Methylen, 
chemisch  rein  und  chlorzinkfrei/'  It  is  therefore  highly 
desirable,  when  ordering  methylen  blue,  to  specify  for  what 
purpose  it  is  required. 

322.  The  Uses  of  Methylen  Blue. — As  a  histological  reagent 
it  is  used  for  sections  of  hardened  central  nervous  tissue,  in 
which  it  gives  a  specific  stain  of  medullated  nerves  ( post, 
Part  II).  It  is  a  valuable  specific  reagent  for  plasma-cells 
(for  which  see  also  Part  II).  In  stains  a  large  number  of 
tissues  intra  vitam,  with  little  or  no  interference  with  their 
vital  functions.  And  last,  not  least,  it  can  be  made  to 
furnish  stains  of  nerve  tissue,  intercellular  cement  substances, 
lymph  spaces,  and  the  like,  that  are  essentially  identical  with 
those  furnished  by  a  successful  impregnation  with  gold  or 
silver,  and  are  obtained  with  greater  ease  and  certainty  ; 
with  this  difference,  however,  that  gold  stains  a  larger 
number  of  the  nervous  elements  that  are  present  in  a 
preparation,  sometimes  the  totality  of  them  ;  whilst  methylen 
blue  stains  only  a  selection  of  them,  so  bringing  them  more 
prominently  before  the  eye,  and  allowing  them  to  be  traced 
for  greater  distances.  So  that  in  this  respect  methylen  blue 
behaves  more  like  Grolgi's  chrome-silver  impregnation. 

323.  Staining  in  toto  during  Life. — Small  and  permeable  aquatic 
organisms  may  be  stained  during  life  by  adding  to  the  water  in  which  they 
are  confined  enough  methylen  blue  to  give  it  a  very  light  tint.  If  trans- 
parent organisms  be  taken,  they  may  be  examined  alive  without  further 
manipulation  at  any  desired  moment,  and  will  be  found  after  a  time  to  be 
partially  stained — that  is,  it  will  be  found  that  certain  tissues  have  taken 
up  the  colour,  others  remaining  colourless.  If  now  you  put  back  the  animals 
into  the  tinted  water  and  wait,  you  will  find  on  examination  after  a  sufficient 
lapse  of  time  that  further  groups  of  tissues  have  become  stained.  Thus  it 
was  found  by  EHELICH  (Biol.  Centralb.,  vi,  1886,  p.  214 ;  Abh.  k.  Akad. 
Wiss.  Berlin,  February  25,  1885),  to  whom  the  principle  of  this  method  is 
due,  that  on  injection  of  the  colour  into  living  animals  axis-cylinders  of 
sensory  nerves  stain,  whilst  motor  nerves  remain  colourless.  [The  motor 
nerves,  however,  will  also  stain,  though  later  than  the  sensory  nerves.]  It 
might  be  supposed  that  by  continuing  the  staining  for  a  sufficient  time,  a 
point  would  be  arrived  at  at  which  all  the  tissues  would  be  found  to  be 
stained.  This,  however,  is  not  the  case.  It  is  always  found  that  the  stained 
tissues  only  keep  the  colour  that  they  have  taken  up  for  a  short  time  after 
they  have  attained  the  maximum  degree  of  coloration  of  which  they  are 


230  CHAPTER   XVII. 

susceptible ;  as  soon  as  that  point  is  attained  they  begin  to  discharge  the 
colour  even  more  quickly  than  they  took  it  up.  And  it  is  very  often  found 
that  the  elements  which  have  stained  first  will  have  lost  much  or  all  of  their 
colour  by  the  time  that  tliose  which  stain  later  have  attained  their  maximum 
coloration.  It  may  even  happen,  as  I  have  observed,  that  the  whole  of  the 
stainable  tissues  of  an  animal  may  run  through  the  total  gamut  of  colora- 
tion and  decoloration  until  the  animal  has  become  as  colourless  as  when 
first  put  into  the  tinted  water,  and  that  without  any  apparent  change  in  its 
vital  activities.  The  stain,  therefore,  that  has  thus  been  produced  and  lost 
is  not  a  true  histological  stain,  but  a  quasi-stain  (see  §  201). 

It  follows  that  a  total  stain  of  all  the  tissues  of  an  organism  can  hardly 
be  obtained  under  these  conditions,  but  that  a  specific  stain  of  one  group  or 
another  of  elements  may  be  obtained  in  one  of  two  ways.  If  the  tissue  to 
be  studied  be  one  that  stains  earlier  than  the  others,  it  may  be  studied 
during  life  at  the  period  at  which  it  alone  has  attained  the  desired  intensity 
of  coloration,  and  the  remaining  tissues  are  not  yet  coloured  at  all,  or  not 
coloured  enough  to  be  an  obstacle  to  observation.  If  it  be  one  that  stains 
later  than  the  others,  it  may  be  studied  during  life  at  the  period  at  which 
the  earlier  stained  elements  have  already  passed  their  point  of  maximum 
coloration  and  have  become  sufficiently  decoloured ;  the  later  stained  ones 
being  at  a  point  of  desired  intensity.  Or  the  observer  may  fix  the  stain  in 
either  of  these  stages  and  preserve  it  for  leisurely  study  by  means  of  one  of 
the  processes  given  below  under  the  heading  "  Preservation  of  the  Prepa- 
rations." 

The  proper  strength  of  the  very  dilute  solutions  to  be  employed  in  the 
manner  here  considered  must  be  made  out  by  experiment  for  each  object.  I 
think  the  tint  is  practically  a  sufficient  guide,  but  it  may  be  stated  that  when 
in  doubt  a  strength  of  1 : 100,000  may  be  taken,  and  increased  or  diminished 
as  occasion  may  seem  to  require.  ZOJA  (Rendic.  R.  1st.  Lombardo,  xxv, 
1892 ;  Zeit.f.  wiss.  MiJc.,  ix,  2, 1892,  p.  208)  finds  that  for  Hydra  the  right 
strength  is  from  1 :  20,000  to  1 : 10,000. 

The  stain  is  capricious.  It  is  not  possible  to  predict  without  trial  which 
tissues  will  stain  first  in  any  organism.  It  is  to  be  remarked  that  the  stain 
penetrates  very  badly,  which  is  perhaps  the  chief  cause  of  its  capriciousness, 
and,  I  take  it,  the  chief  determining  condition  of  the  order  in  which  tissues 
stain.  Gland  cells  generally  stain  early;  then,  in  no  definable  order,  other 
epithelium  cells,  fat  cells,  plasma  cells,  "  Mastzellen,"  blood  and  lymph 
corpuscles,  elastic  fibres,  smooth  muscle,  striated  muscle.  There  are  other 
elements  that  stain  in  the  living  state,  but  not  when  the  staining  is  per- 
formed by  simple  immersion  of  intact  animals  in  a  dilute  staining  solution 
in  the  manner  we  are  considering.  Chief  amongst  these  are  nerve-fibres 
and  ganglion  cells,  which  remain  unstained  in  the  intact  organism, — most 
likely,  so  far  as  I  can  see,  for  the  simple  reason  that  the  stain  is  not  able  to 
penetrate  them. 

324.  Staining  Nervous  Tissue  during  Life. — As  stated  above, 
it  was  made  out  by  EHRLICH  that  by  injecting  a  solution  of 
methyl  en  blue  into  the  vessels  or  tissues  of  living  animals 


METHYLEN    BLUE.  231 

there  may  be  obtained  a  specific  stain  of  axis-cylinders  of 
sensory  nerves.  He  held,  and  it  has  long  been  held  by 
observers,  that  the  stain  so  obtained  is  a  product  of  a  vital 
reaction  of  the  tissues,  and  that  it  cannot  be  obtained  with 
dead  material.  From  the  point  of  view  maintained  in  §  201 
the  contrary  would  be  the  real  state  of  the  case.  The  stain 
is,  of  course,  an  intra-vitam  phenomenon,  in  so  far  as  it  takes 
place  during  the  life  of  the  organism  ;  but  I  hold  that  the 
tissue  itself  does  not  take  on  the  stain  till  it  is  dead  or  patho- 
logically affected. 

As  said  before,  it  was  formerly  held  that  the  reaction 
could  not  be  obtained  with  dead  material.  DOGIEJ.,  however 
(Arch.f.  milf.Anat.j  xxxv,  1890^  pp.  305  et  seq.),  found  that 
muscle  nerves  of  limbs  of  the  frog  could  be  stained  as  much 
as  from  three  to  eight  days  after  the  limbs  had  been  removed 
from  the  animal.  He  concludes,  indeed,  that  the  reaction 
shows  that  the  nerves  were  still  living  at  that  time.  But  it- 
seems  more  natural  to  conclude  with  APATHY  (Zeit.  f.  wis*. 
A/7A\,  ix,  1,  1892,  pp.  15  et  seq.)  that  nerve  tissue  can  bo 
stained  after  life  has  ceased. 

APATHY  has  directly  experimented  on  this  point,  and  sums 
up  the  necessary  conditions  as  follows  : — The  tissue  need  not 
be  living,  but  must  be  fresh ;  nothing  must  have  been 
extracted  from  it  chemically,  and  its  natural  state  must  not 
have  been  essentially  changed  by  physical  means.  For 
example,  the  tissue  must  not  have  been  treated  with  even 
dilute  glycerin,  nor  with  alcohol,  though  a  treatment  for  a 
short  time  with  physiological  salt  solution  is  not  very  hurtful ; 
it  must  not  have  been  coagulated  by  heat. 

Another  common  belief  concerning  the  methylen-blue 
nerve  reaction  is  that  the  presence  of  oxygen  is  necessary  to 
the  reaction.  It  is,  therefore,  the  usual  practice  to  dissect 
out  the  organ  to  be  investigated  after  having  exposed  it  to 
the  action  of  methylen  blue  by  injection  or  immersion,  and 
leave  it  for  some  time  exposed  to  the  air.  AI'ATHY  has  also 
investigated  this  point,  and  finds  (/or.  cit.,  p.  25)  that  the 
practice  is  in  some  cases  correct,  but  the  belief  erroneous. 
His  point  of  view  is  that  the  stain  is  a  regressive  one.  It  has 
been  explained  above  that  shortly  after  a  tissue  has  attained 
the  maximum  degree  of  coloration  of  which  it  is  susceptible 
it  begins  to  give  up  its  colour  again  to  the  surrounding  liquid. 


232  CHAPTER   XVII. 

The  larger  the  volume  of  liquid  with  which  the  tissue  is  sur- 
rounded, the  faster  will  this  washing-out  process  go  on ;  and 
in  order  that  it  may  not  go  on  with  excessive  rapidity,  wash- 
ing out  the  stain  from  the  nerve-fibres  as  well  as  from  the 
earlier  stained  elements  (which  alone  it  is  desired  to  wash 
out,  so  as  to  leave  a  differentiated  specific  stain  of  nervous 
elements),  it  is  in  many  cases  desirable  to  have  the  process 
go  on  in  presence  of  as  little  liquid  as  possible.  Another 
consideration  that  justifies  the  practice  is  that  by  exposure  to 
air  the  preparations  take  up  a  trace  of  ammonia,  and  APATHY 
has  experimentally  established  that  this  is  an  important  factor 
in  the  sharpness  of  the  stain.  Oxygen  has,  according  to 
him,  nothing  to  do  with  it.  But  the  point  does  not  seem  to 
be  definitively  settled.  RUBASCHKJN  (see  Zeit.  f.  wiss.  Milt., 
xvi,  3,,  1899,  p.  372)  has  lately  concluded  that  the  staining 
of  nerve-fibres  (not  of  all  tissues)  is  furthered  by  oxygen,  and 
also  by  CO2. 

325.  Staining  Nerve  Tissue  by  Injection  or  Immersion — The 

practice  of  the  earlier  workers  at  this  subject  was  (following 
EHKLICH)  to  inject  methylen  blue  into  the  vascular  system  or 
body-cavity  of  a  living  animal,  wait  a  sufficient  time  for  it  to 
take  effect  on  the  organ  to  be  stained,  then  remove  the  organ 
for  further  preparation  and  study.  And  there  appears  to 
have  been  a  belief  with  some  workers  that  it  was  an  essential 
or  at  least  a  desirable  condition  to  the  production  of  the  stain 
that  it  should  have  been  brought  about  by  injection  of  the 
colouring  matter  into  the  entire  animal.  It  is  now  known 
that  this  is  generally  immaterial,  and  that  the  reaction  can 
equally  well  be  obtained  by  removing  the  organ  and  subject- 
ing it  to  a  bath  of  the  colouring  matter  in  the  usual  way. 
But  it  would  also  appear  that  in  some  cases  the  procedure 
by  injection  is  preferable,  if  not  necessary. 

326.  The  Solutions    employed. — The   solutions  used  for  in- 
jection are  generally  made  in  salt  solution  (physiological,  or  a 
little  weaker)  ;  those  for  staining  by  immersion  either  in  salt 
solution   or    other    "  indifferent "   liquid,    or   in  pure  water. 
Very  various   strengths  have  been  employed.      The    earlier 
workers     generally     took     concentrated     solutions.        Thug 
ARNSTEIN  (Anat.  Anz.}  1887,  p.  125)   injected  1  c.c.  of   satu- 


METHYLEN    BLUE.  233 

rated  (i.  e.  about  4  per  cent.)  solution  into  the  vena  cutanea 
magna  of  frogs,  and  removed  the  organ  to  be  investigated 
after  the  lapse  of  an  hour.  BIEDERMANN  (Sitzb.  d.  k.  Akad. 
MV.s-x.  Wien,  Math.  Nat.  CL,  1888,  p.  8)  injected  0'5  to  1  c.c. 
of  a  nearly  saturated  solution  in  0'6  per  cent,  salt  solution 
into  the  thorax  of  crayfishes,  and  left  the  animals  for  from 
two  to  four  hours  before  killing  them.  S.  MAYER  (Zeit.  f. 
•tcis*.  If /A-.,  vi,  4,  1880,  p.  423)  took  a  strength  of  1  :  300  or 
400  of  0*5  per  cent,  salt  solution.  This  can  be  introduced 
into  the  system  either  by  means  of  a  syringe  or  other  inject- 
ing apparatus,  or  by  auto -injection  through  the  heart.  Even 
rabbits  support  this  operation  if  artificial  respiration  be  main- 
tained. The  solutions  of  RETZIUS  are  of  the  same  strength. 
But  the  tendency  of  more  recent  practice  is  decidedly  towards 
the  employment  of  weaker  solutions.  APATHY  (Zeit.f.  wis*. 
J//A-.,  ix,  1,  1892,  pp.  25,  26,  et  *eq.)  finds  that  it  is  not  only 
superfluous,  but  positively  disadvantageous,  to  take  solutions 
stronger  than  1  :  1000. 

For  staining  by  immersion  similar  solutions  to  those  used 
for  injecting  may  be  employed,  but  they  should,  if  anything, 
be  still  weaker.  DOGIKL  (Arch.  f.  mik.  Anat.,  xxxv,  1890, 
p.  305  ;  Zeit.  f.  u-iss.  Mik.,  vii,  4,  1891,  p.  509)  places  objects 
in  a  few  drops  of  aqueous  or  vitieous  humour,  to  which  are 
added  two  or  three  drops  of  a  -^  to  y-  per  cent,  solution  of 
methylen  blue  in  physiological  (0*75  percent.)  salt  solution,  and 
exposes  them  therein  to  the  air.  In  thin  pieces  of  tissue  the 
stain  begins  to  take  effect  in  five  or  ten  minutes,  and  attains 
its  maximum  in  from  fifteen  to  twenty  minutes.  For  thicker 
specimens — retina,  for  instance — several  hours  may  be  neces- 
sary, the  preparation  being  kept  just  moist  by  occasional 
treatment  with  a  drop  or  two  of  indifferent  liquid  or  methylen 
blue  solution,  added  by  turns.  The  reaction  is  quickened  by 
putting  the  preparations  into  a  stove  kept  at  30°  to  35°  C. 
ROUQET  (Gompt.  Rend.,  1893,  p.  802)  found  it  useful  to 
modify  the  procedure  of  Dogiel  by  employing  a  00' 5  per 
cent,  solution  in  0'6  per  cent,  salt  solution  (for  muscles  of 
Batrachia).  ALLKN  (Quart.  Journ.  3//c.  Sci.,  1894,  pp.  461, 
483)  takes  for  embryos  of  the  lobster  a  solution  of  O'l  per 
cent,  in  0'75  per  cent,  salt  solution,  and  dilutes  it  with  15 
to  20  volumes  of  sea  water. 


234  CHAPTER  XVII. 

327.  APATHY'S  Methods. — As  a  good  example  of  this  kind 
of  work,  I  subjoin  a  short  account  of  the  procedure  recom- 
mended by  Apathy  (Zeit.  f.  wins.  Mik.,  ix,  1,  1892,  p.  15  ; 
see  also  his  Mikrotechnilc,  p.  172)  for  Hirudinea.  A  portion 
of  the  ventral  cord  is  exposed,  and  if  it  be  considered  desir- 
able dissected  out,  but  the  sinus  and  pigmented  connective 
tissue  around  it  had  better  not  be  removed  till  the  staining* 
and  fixation  are  completed.  If,  however,  it  be  desired  to 
stain  as  many  ganglion  ceils  as  possible,  as  well  as  fibres,  the 
lateral  nerves,  as  well  as  the  connectives,  should  be  cut 
through  near  a  ganglion.  The  preparation  is  then  treated 
with  the  stain.  This  is,  for  the  demonstration  chiefly  of 
fibres  in  Htrudo  and  Puntobdeila,  either  a  1  :  1000  solution 
in  0'5  to  0*75  per  cent,  salt  solution,  allowed  to  act  for  ten 
minutes  ;  or  a  1  :  10,000  solution  allowed  to  act  for  an  hour 
to  an  hour  and  a  half ;  or  a  1  :  100,000  solution  allowed  to 
act  for  three  hours  (Liunbric-uts  requires  twice  these  times  ; 
Antaeus  and  Unio  require  three  times  ;  medullated  nerves  of 
Vertebrates  four  times) .  For  the  demonstration  of  ganglion 
cells  the  stain  is  allowed  to  act  three  or  four  times  as  long. 

The  staining  having  been  accomplished,  the  preparations 
from  the  1  :  1000  solution  are  washed  in  salt  solution  for  an 
hour  ;  those  from  the  1  :  10,000  solution  for  a  quarter  of  an 
hour;  those  from  the  1  :  100,000  solution  need  not  be 
washed  at  all.  They  are  then  treated  with  one  of  the  am- 
moniacal  fixing  and  differentiating  liquids  described  below  in 
the  next  section.  This  is  done  by  pouring  the  liquid  over 
them,  and  leaving  them  in  it  without  'moving  them  about  in  it 
for  at  least  an  hour,  and  by  preference  in  the  dark.  The 
further  treatment  is  as  described  in  the  next  section. 

The  object  of  the  ammonia  in  these  liquids  is  to  differentiate 
the  stain — to  produce  an  artificial  "  secondary  differentiation." 
It  acts  by  washing  out  the  absorbed  colour  from  certain 
elements,  others  resisting  its  action  longer,  much  as  HC1 
alcohol  washes  out  a  borax- carmine  stain.  In  this  case  the 
elements  that  are  Avashed  out  are  the  protoplasmic  parts  of 
nerve-fibres,  and  their  "  interfibrillar  "  and  "  perifibrillar  " 
substance,  the  "primitive  fibrils"  still  retaining  the  colour 
strongly.  It  is  of  theoretical  interest  to  remark  that  accord- 
ing to  APATHY  the  coloration  thus  obtained  is  a  true  xtain  of 
the  "  primitive  fibrils/'  not  an  impregnation.  The  "  primitive 


METHYLEN    BLUE.  235 

fibrils "  are  sharply  stained  of  a  violet-blue,  showing  no 
granular  precipitate,  and  the  "  interfibrillar "  and  "  peri- 
fibrillar  "  substance,  as  well  as  nuclei,  are  either  colourless 
or  very  lightly  stained.  The  usual  methods,  on  the  other 
hand,  give  an  "  inverse  "  reaction,  the  "  primitive  fibrils  " 
remaining  colourless,  whilst  the  interfibrillar  substance  and 
protoplasm  of  the  nerve-fibres  are  impregnated  with  a  finely 
granular  greenish -black  or  violet  precipitate,  nnd  the  nuclei 
are  usually  stained. 

328.  Preservation  of  the  Preparations. — There  are  consider- 
able difficulties  in  the  way  of  obtaining  permanent  prepara- 
tions, as  the  stain  is  so  very  unstable  that,  as  above  explained, 
it  begins  to  discharge  after  a  short  time,  even  in  the  living 
and  not  yet  totally  impregnated  tissue.  It  may,  however, 
be  fixed,  and  more  or  less  permanent  preparations  be  made 
by  one  or  other  of  the  following  methods  : 

DOGIEL  (Arch.f.  m-ili'.  Anat.,  xxxiii,  4,  1889,  pp.  440  et  *eq.}9 
following  AKNSTEIN  (AnQt.Au.zeig.,  1887,  p.  551),  brings  the 
preparations,  in  order  to  fix  the  colour,  into  saturated  aqueous 
solution  of  picrate  of  ammonia,  in  which  they  are  allowed  to 
remain  for  half  an  hour  or  more,  and  are  then  removed, 
washed  in  fresh  picrate  of  ammonia  solution,  and  studied  in 
dilute  glycerine,  or  mounted  permanently  in  glycerine  satu- 
rated with  picrate  of  ammonia.  More  recently  (Zeit.  f.  ic-is*. 
Af/fc.,  viii,  1,  1891,  p.  15)  he  has  recommended  an  increased 
duration  of  the  picrate  of  ammonia  bath  up  to  eighteen  or 
twenty-four  hours,  and  mounting,  without  washing  out,  in 
chemically  pure  glycerin,  free  from  acid.  There  is  a  defect 
in  this  process,  namely,  that  picrate  of  ammonia  has  a  very 
injurious  action,  of  a  macerating  nature,  on  some  tissues. 
This  may,  however,  be  avoided  by  adding  to  the  fixing-bath 
I  to  2  per  cent,  of  a  1  per  cent,  osmic  acid  solution.  (If  it- 
be  desired  to  harden  the  tissues  so  that  sections  ma}'  be  cut, 
the  proportion  of  osmium  solution  should  be  increased  four- 
fold.) 

S.  MAYER  (Zeit.  f.  wiss.  Mik.,  vi,  4,  1889,  p.  422)  preferred 
a  mixture  of  equal  parts  of  glycerin  and  saturated  picrate 
of  ammonia  solution,  which  served  to  fix  the  colour  and 
mount  the  preparations  in.  This  was  also  in  principle  the 
method  followed  by  RETZIUS  (Intern.  Monatsschr.  Anat.  u. 


236  CHAPTER   XVII. 

Phys.,  Bd.  vii,  H.  8,  1890,  p.  328).  DOGIEL,  after  careful 
study,  quite  refuses  to  admit  that  this  is  in  any  way  an  im- 
provement. 

Other  workers  have  employed  saturated  solution  of  iodine 
in  iodide  of  potassium  (so  ARNSTETN)  or  picro-carmine  (so 
FEIST,  Arch.  f.  Anat.  u.  Entwickel.,  1890,  p.  116;  cf.  Zeit.  f. 
wiss.  Mik.,  vii,  2,  1890,  p.  231),  the  latter  having  the  advan- 
tage of  preserving  the  true  blue  of  the  stain  if  it  be  not 
allowed  to  act  too  long,  and  the  preparation  be  mounted  in 
pure  glycerin. 

Picric  acid  has  been  used  by  LAVDOWSKY,  but  this  too,  after 
careful  study,  is  rejected  by  DOG i EL. 

APATHY  (Zeit.f.  wiss.  Mik.,  ix,  1,  1892,  p.  30)  has  found,  as 
stated  above,  that  free  ammonia  is  a  capital  factor  in  the 
differentiation  of  the  stain.  He  brings  preparations  (after 
washing  in  salt  solution  if  the  staining  have  been  performed 
with  a  strong  methylen  blue  solution,  or  without  washing  if 
it  have  been  done  with  a  very  dilute  solution)  either  into  a 
concentrated  aqueous  solution  of  picrate  of  ammonia  free 
from  picric  acid,  and  containing  five  drops  of  concentrated 
ammonia  for  every  100  c.c.  ;  or,  which  is  generally  prefer- 
able, into  a  1  to  2  per  cent,  freshly  prepared  solution  of 
neutral  carbonate  of  ammonia  saturated  with  picrate.  They 
remain  in  either  of  these  solutions,  preferably  in  the  dark, 
for  at  lea$t  an  hour.  They  are  then  brought  into  a  small 
quantity  of  saturated  solution  of  picrate  of  ammonia  in  50 
per  cent,  glycerin,  where  they  remain  until  thoroughly 
saturated.  They  are  then  removed  into  a  saturated  solution 
of  the  picrate  in  a  mixture  of  2  parts  50  per  cent,  glycerin, 
1  part  cold  saturated  sugar  solution,  and  1  pnrt  similarly 
prepared  gum-arabic  solution.  When  thoroughly  penetrated 
with  this  they  are  removed  and  mounted  in  the  following 
gum-syrup  medium  (loc.  cit.,  p.  37)  : 

Picked  gum-arabic  .  .  .50  grms. 

Cane-sugar  (not  candied)  .  .      50      ,, 

Distilled  water       .  .  .  50      „ 

Dissolve  over  a  water-bath  and  add  0'05  grm.  thymol.      This 

mounting  medium  sets   quickly  and  as  hard  as   balsam,    so 

that    no    cementing  of  the  mounts  is  necessary.      Fax-rants' 

medium  (with  omission  of  the  arsenious  acid)  will  also  do. 


METHYLEN    BLUE.  237 

In  neither  case  should  either  ammonium  picrate  or  methylen 
blue  be  added  to  the  medium. 

Preparations  that  have  been  fully  differentiated  by  ammonia  do  not  keep 
more  than  a  fe\v  weeks;  whilst  those  in  which  the  differentiation  has  not 
been  carried  to  the  point  of  thorough  tinctorial  isolation  of  the  "neuro- 
tibrils"  have  kept  for  five  or  six  years  (APATHY,  Mitth.  ZooL  Stat.  Neapel, 
xii,  1S97,  p.  712). 

Preparations  preserved  by  these  methods  (I  do  not  know 
whether  it  is  the  case  with  preparations  preserved  by  PARKER'S 
or  BETHE'S  method,  next  section)  are  extremely  sensitive  to- 
the  influence  of  light.  APATHY  finds  that  lamplight  is  par- 
ticularly injurious,  especially  the  intense  lamplight  used 
with  high  powers. 


329.  Methods  for  Sections. — None  of  the  preceding  methods 
can  be  said  to  be  anything  like  perfectly  satisfactory.  They 
do  not  give  preparations  that  will  resist  the  operations 
necessary  for  imbedding  in  paraffin.  The  stain  is  generally 
not  preserved  in  its  true  blue  colour,  but  turns  to  a  grey, 
varying  in  tone  from  reddish  browi*  to  bluish  or  greenish 
black.  The  preparations  seldom  keep  even  in  that  state  for 
more  than  a  very  few  months,  and  it  is  not  satisfactory  to  be 
obliged  to  mount  preparations  only  in  aqueous  media.  A 
strong  solution  of  platinum  chloride  is  said  to  give  a  fixation 
that  will  resist  the  treatment  necessary  for  imbedding  either 
in  celloidin  or  paraffin  (see  FEIST,  Arch.  f.  Anat.  und  Entw., 
1890,  p.  116  ;  Zeit.  f.  wixs.  Mik.,  vii,  2,  1890,  p.  231)  ;  but 
the  precipitate  it  gives  is  a  flocculent  one,  and  the  prepara- 
tions are  not  very  satisfactory. 

.For  the  earlier  method  of  PARKER  (Zool.  Anzeig.,  No.  403, 
1892,  p.  375)  with  methylal  see  previous  editions.  Later 
(Mitth.  ZooL  Stat.  Neapel,  xii,  1895,  p.  4)  he  fixes  the  stain 
by  dehydrating  the  objects  in  successive  alcohols  of  30,  50, 
70,  95,  and  100  per  cent,  strength,  each  containing  8  per 
cent,  of  corrosive  sublimate,  then  brings  them  into  a  mixture 
of  the  last  with  an  equal  volume  of  xylol,  and  lastly  into 
pure  xylol. 

The  method  of  BETHE  (Arch.  f.  mik.  Anat.,  xliv,  1894, 
p.  585),  in  its  first  form,  is  as  follows  : — A  solution  is  made 
of— 


238  CHAPTER   XVII. 

Molybdate  of  ammonium  .  .  1  grm. 

Distilled  water          .  .  .  .10  grms. 

Peroxide  of  hydrogen        ...  1  grm. 

On  adding  the  peroxide  a  yellow  colour  is  produced.  A 
drop  of  hydrochloric  acid  is  added  (white  precipitate  of 
molybdic  acid  which  dissolves  on  agitation).  After  staining 
and  rinsing  in  salt  solution  the  preparations  are  put  into  the 
molybdic  solution.  The  solution  ought  not  to  be  more  than 
eight  days  old,  and  it  is  well  to  use  it  cooled  to  zero.  The 
preparations  are  left  in  it  for  two  or  three  hours  if  they  are 
small,  or  four  or  five  hours  if  they  are  large  (of  a  centimetre 
in  size) .  They  are  then  washed  for  from  half  an  hour  to  two 
hours  in  water,  dehydrated  in  alcohol  (which  it  is  well  to  use 
cooled  to  zero),  and  cleared  in  clove  oil,,  or,  better,  in  xylol. 
They  may  then  be  imbedded  either  in  paraffin  or  celloidin  in 
the  usual  way. 

This  is  for  tissues  of  Vertebrates.  For  Invertebrates 
BETHE  takes  one  grm.  of  molybdate,  10  c.c.  of  water,  and  0'5 
<?.c.  of  peroxide. 

PEABODY  (Zool.  Bull,  i,  1897,  p.  163;  Zeit.f.  iviss.  Mile.,  xvi,  1,  1899, 
p.  73)  adds  a  drop  of  1  per  cent,  osinic  acid. 


ETHK'S  later  method  (Anat.  Ariz.,  xii,  1896,  p.  438)  is  as 
follows  : — After  staining,  pieces  of  tissue  of  2  to  3  mm.  thick- 
ness are  treated  for  ten  to  fifteen  minutes  with  a  concentrated 
aqueous  solution  of  picrate  of  ammonia  and  are  then  brought 
into  a  solution  of  1  grm.  of  molybdate  of  ammonium,  either 
in  20  of  water,  or  in  10  of  water  and  10  of  0*5  per  cent, 
osmic  acid  or  2  per  cent,  chromic  acid  ;  or  into  a  solution  of 
phosphomolybdate  of  sodium  in  the  same  proportions,  each 
of  these  solutions  having  added  to  it  1  drop  of  hydrochloric 
acid,  and  if  desired  1  grm.  of  peroxide  of  hydrogen.  They 
remain  in  one  of  these  solutions  for  three  quarters  to  one  hour 
(or  from  four  hours  to  twelve  in  the  osmic  acid  one),  and  are 
then  passed  through  water,  alcohol,  xylol,  balsam,  or  paraffin. 
(The  objects  that  have  been  treated  with  one  of  the  solutions 
of  the  sodium  salt  are  not  thoroughly  resistent  to  alcohol,  so 
that  for  them  it  is  well  to  cool  the  alcohol  to  under  15°  C.) 
Sections  may  be  after-stained  with  alum  carmine,  or 
"  neutral  "  tar  colours. 

Slight  modifications  of  this  method  are  given  by  DOGIEL 


METHYLEN    BLUE.  239 

(Arch.  mik.  An  at.,  xlix,    1897,   p.  772;   liii,    1898,  p.   237; 
and  Zeit.f.  wis*.  Zool.,  Ixvi,  1899,  p.  361). 

PLESCHKO  (Anat.  Am.,  xiii,  1897,  p.  16)  fixes  with  picrate  of  ammonia, 
and  then  puts  into  10  per  cent,  formol  for  a  few  days. 

HARRIS  (Philadelphia  Medical  Journ.,  May  14th,  1898) 
after  staining  rinses  with  water,  and  brings  the  specimens 
into  a  saturated  solution  of  either  ferrocyanide  or  ferri- 
cyanide  of  potassium  which  has  been  cooled  to  within  a  few 
degrees  of  zero  (a  trace  of  osmic  acid  may  be  added  to  prevent 
maceration).  They  remain  therein  for  three  to  twenty-four 
hours,  and  are  then  washed  in  distilled  water  for  an  hour,  and 
are  dehydrated  in  absolute  alcohol  kept  at  a  low  temperature, 
cleared  in  xylol  or  cedar  oil,  and  imbedded  in  paraffin. 

330.  Methylen  Blue  Impregnation  of  Epithelia,  Lymph-spaces, 
etc.  (DOGIEL,  Arch.f.  mik.  An  at.,  xxxiii,  4,  1889,  p.  440,  et  seq.). 
—  Suitable  pieces  of  tissue  (thin  membranes  by  preference) 
are  brought  fresh  into  a  4  per  cent,  solution  of  methylen 
blue  in  physiological  salt  solution.  After  a  few  minutes 
therein  they  are  brought  into  saturated  solution  of  picrate  of 
ammonia,  soaked  therein  for  half  an  hour  or  more,  then 
washed  in  fresh  picrate  of  ammonia  solution,  and  examined 
in  dilute  glycerin. 

If  it  be  wished  only  to  demonstrate  the  outlines  of  endo- 
thelium  cells,  the  bath  in  the  stain  should  be  a  short  one,  not 
longer  than  ten  minutes  in  general  ;  whilst  if  it  be  desired 
to  obtain  an  impregnation  of  ground-substance  of  tissue,  so 
as  to  have  a  negative  image  of  juice  canals  or  other  spaces, 
the  staining  should  be  prolonged  to  fifteen  or  thirty  minutes, 
and  it  is  advisable  to  remove  the  endothelial  covering  of  the 
objects  operated  on  before  putting  them  into  the  stain. 

If  it  be  desired  to  preserve  the  preparations  permanently, 
they  had  better  be  mounted  in  glycerin  saturated  with  picrate 
of  ammonia.  (For  an  improvement  in  the  method  of  pre- 
servation given  in  a  later  paper  see  supra,  §  328.) 

The  effect  is  practically  identical  (except  as  regards  the 
colour)  with  that  of  a  negative  impregnation  with  silver 
nitrate. 


331.  MAYER'S  (S.)  Impregnation  Methods  (Zeit.f.  wiss. 
vi,  4,  1889,  p.  422).  —  MAYER'S  experiments  cover  much  the 


240  CHAPTER    XVII. 

same  ground  as  those  of  DOGIEL,  and  give  the  same  results. 
MAYEK  stained  tissues  for  about  ten  minutes  in  a  1  :  300  or 
400  solution  of  methylen  blue  in  0*5  per  cent,  salt  solution, 
rinsed  in  salt  solution,  and  put  up  in  the  glycerin -picrate  of 
ammonia  mixture  given  above,  §  328.  He  finds  that  by  this 
method  all  the  essential  result*  of  a  nitrate  of  silver  impreg- 
nation may  be  produced  by  means  of  methylen  blue.  The 
images  are  either  positive  or  negative,  as  with  silver.  If  the 
stain  be  brought  about  by  injection  of  the  colour  into  the  vas- 
cular system,  the  positive  impregnation  is  the  more  frequent ; 
whilst  if  it  have  been  brought  about  by  the  immersion  of  the 
tissues  (cornea),  a  negative  image  is  more  frequently 
obtained. 

332.  Toluidm  Blue  or  Thionin  as  succedanea  of  methylen 
blue. — HAKRIS  (Philadelphia  Med.  Journ.,  May  14th,  1898) 
has  found  that  there  is  no  reaction  of  methylen  blue  that 
cannot  be  equally  well  obtained  with  toluidin  blue  or  thionin. 
Indeed,  he  thinks  that  for  nerves  they  may  have  some  points 
of  superiority.  For  staining  pieces  of  tissue  he  recommends 
the  following  : 

Toluidm  blue,   O'l   per   cent.    sol.    in 

physiological  salt  solution       .  .  2  parts 

Ammonium  chloride,  0'25  per  cent,  in 

water  .....  1  part 

Egg  albumen  .  .  .  .  1      „ 

For  injections  he  uses  1  part  of  the  dye  to  1000  of  physio- 
logical salt  solution. 

Any  of  the  methylen  blue  fixing  methods  may  be  employed, 
and  the  whole  technique  is  the  same.  Harris's  fixing  method 
has  been  given,  §  329. 


CHAPTER  XVIII. 

METALLIC   STAINS    (IMPREGNATION   METHODS). 

333.  The  Characters  of  Impregnation  Stains. — By  impregna- 
tion is  understood  a  mode  of  coloration  in  which  a  colouring" 
matter  is  deposited  in  certain  elements  of  tissues  in  the  form 
of  a  more  or  less  finely  granular  or  sometimes  even  flocculent 
precipitate — the   impregnated   elements   becoming  in  conse- 
quence opaque.      By  staining,,  on  the  other  hand,  is  under- 
stood a  mode  of  coloration  in  which  the  colouring  matter  is 
retained  by  the  tissues  as  if  in  a  state  of  solution,  showing 
110  visible   solid  particles  under  the  microscope,  the  stained 
elements   remaining    in    consequence    transparent.       But    it 
should  be  understood  that  it  is  not  correct  to  draw  a  hard 
and  fast  line  between  the  two  kinds  of  coloration.      Some  of 
the  metallic  salts  treated  of  in  this  chapter  give,  besides  an 
impregnation,  in  some  cases  a  true  stain.      And  some  of  the 
dyes  that  have  been  treated  of  in  preceding  chapters  give, 
besides  a   stain,  a  true  impregnation.      Methylen  blue,   for 
instance,  will  give  in  one  and  the  same  preparation  an  im- 
pregnation and  a  stain;    and  critical  examination  of    most 
fairly  successful   gold   chloride  preparations  will  show  that 
the  coloration  is  in  places  of  the  nature  of  a  finely  divided 
solid  deposit,  in  others  a  perfectly  transparent  stain. 

Thus  is  justified  the  alternative  title  that  this  chapter  has 
always  borne — Metallic  Stains,  or  Impregnation  Methods. 

334.  Negative  and  Positive  Impregnations. — Impregnations 
are  distinguished  as   negative  and  positive.      In  a  negative 
impregnation  intercellular  substances  alone  are  coloured,  the 
cells  themselves  remaining  colourless  or  very  lightly  tinted. 
In   a   positive   impregnation  the   cells   are   stained   and  the 
intercellular  spaces  are  unstained.      (This  explanation  is  the 

16 


242  CHAPTER  XVIII. 

more  needful  as  a  directly  contrary  statement  is  made  in   ;i 
recent  Lehrbuch.) 

Negative  impregnation  is  generally  held  to  be  primary 
because  it  is  brought  about  by  the  direct  reduction  of  a 
metal  in  the  intercellular  spaces.  Positive  impregnation  is 
held  to  be  secondary  (in  the  case  of  silver  nitrate  at  least) 
because  it  is  brought  about  by  the  solution  in  the  liquids  of 
the  tissues  of  the  metallic  deposit  formed  by  a  primary  or 
negative  impregnation,  and  the  consequent  staining  of  the 
cells  by  the  new  solution  of  metallic  salt  thus  formed. 
These  secondary  impregnations  take  place  when  the  reduc- 
tion of  the  metal  in  the  primary  impregnation  is  not  suffi- 
ciently energetic  (see  on  these  points  His,  Scliweizer  Zeit.  f. 
Heilk.,  ii,  Heft  1,  p.  1  ;  G-IKEKK,  Zeit.  f.  wi&s.  Mik.,  i,  p.  393 ; 
HANVIUE,  Traite,  p.  107). 

335.  Nature  of  the  Metallic  Deposit. — As  to  the  nature  of  the  black 
•or  brown  deposit  or  stain  formed  in  the  intercellular  spaces  in  cases  of 
primary  impregnation  see  previous  editions,  or  SCHWALBE,  Arch.  f.  mik. 
Anat.,  vi,  1870,  p.  5;  GIERKE'S  Fdrberei  zu  mikroskopischen  Zwecken,  in 
vols.  i  and  ii  of  Zeit.  f.  wiss.  Mik. ;  JOSEPH,  Sitzb.  d.  k.  prems.  Akad. 
d.  Wiss.  Berlin,  1888 ;  Zeit.  f.  iviss.  Mik.,  xi,  1,  1894,  p.  42,  et  seq. 

336.  Action  of  Light  on  Solutions  of  Metallic  Salts.— Stock 
solutions  of  metallic  salts  are  generally  kept  in  the  dark,,  or 
at  least  in  coloured  bottles,  under  the  belief  that  exposure 
to  light  spoils  them  by  precipitating  the  metal  in  a  state  of 
reduction.  It  has  been  pointed  out  in  §  33  that  in  the  case 
of  osmic  acid  not  light,  but  dust  is  the  reducing  agent ;  and 
that  solutions  may  be  exposed  to  light  with  impunity  if  dust 
be  absolutely  denied  access  to  them.  I  have  now  good 
evidence  to  the^  effect  that  the  same  is  the  case  with  other 
metallic  solutions  ;  and  the  interesting  point  is  raised  whether 
such  solutions  are -not  positively  improved  for  impregnation 
purposes  by  exposure  to  light !  Dr.  LINDSAY  JOHNSON  has 
been  examining  this  question  both  from  a  histological  and 
from  a  photographic  point  of  view,  and  writes  me  as  follows  : 

"  One  may  (I  find  by  experiment)  state  as  a  rule  without 
exception  that  all  the  solutions  of  the  chlorides  and  nitrates 
of  the  metals  will  keep  indefinitely  in  clean  white  stoppered 
bottles  in  the  sunlight ;  and  as  far  as  osmium,  uranium,  gold 
-and  silver,  and  platinum  are  concerned,  actually  improve  or 


MKTALLIC    STAINS    (IMPREGNATION    METHODS).         243 

ripen  by  a  good  sunning.  All  photographers  tell  me  their 
papers  salt  more  evenly  by  old  well-sunned  silver  nitrate 
rhan  by  a  fresh  solution  kept  in  the  dark ;  and  I  go  so  far 
as  to  say  that  this  is  one  of  the  reasons  why  gold  stains  are 
so  unsatisfactory." 

APATHY  (Mitth.  Zool.  Stat.  Neapel,  xii,  1897,  p.  722) 
leaves  his  gold  solutions  exposed  to  light,  so  long  as  there 
are  no  tissues  in  them. 

337.  State  of  the  Tissues  to  be  impregnated.— It   has   been 
pointed  out   in  earlier  chapters  that  the  majority  of  histo- 
logicnl  stains  are   not  obtained  with  fresh  tissues,  but  with 
tissues  that  have  been  more   or  less  charged  with  metallic 
salts,  or  otherwise  changed  in  their  composition  by  the  action 
of  fixing  and  preservative  reagents.      With  metallic  impreg- 
nations the  case  is  different ;  perfectly  fresh  tissues — that  is 
such  as    are   either   living,   or   at  all  events  have  not  been 
treated  by  any  reagent  whatever — will  also  impregnate  with 
the  greatest  ease  and  precision.      Indeed,  some  sorts  of  im- 
pregnations will  not  succeed  at  all  with  tissues  that  are  not 
fresh  in  the  sense  above  explained. 

Silver. 

338.  Silver  Nitrate:    Generalities. — This  is  the  most   com- 
monly  used   salt   of   silver.      The   general   principles   of   its 
employment   are  so  well  stated  by  RANVIER  (Trait*',  p.  105) 
that  I  cannot  do  better  than  abstract  his  account. 

Silver  nitrate  may  be  employed  either  in  solution  or  in 
the  solid  state.  The  latter  method  is  the  less  frequently 
employed,  but  is  easy  and  gives  good  results.  It  is  useful 
for  the  study  of  the  cornea  and  of  fibrous  tissue,  but  is  not 
suitable  for  epithelia.  For  the  cornea,  for  instance,  proceed 
as  follows: — The  eye  having  been  removed,  a  piece  of  silver 
nitrate  is  quickly  rubbed  over  the  anterior  surface  of  the 
cornea,  which  is  then  detached  and  placed  in  distilled 
water ;  it  is  then  brushed  with  a  camel's  hair  brush  in  order 
to  remove  the  epithelium.  The  cornea  is  then  exposed  to 
the  action  of  light.  On  subsequent  examination  it  will  be 
found  that  the  silver  nitrate,  which  was  dissolved  by  the 
liquid  that  bathes  the  surface  of  the  cornea,  has  traversed 


244  CHAPTEE   XVIII. 

the  epithelium  and  soaked  into  the  fibrous  tissue,  on  the 
surface  of  which  it  is  reduced  by  the  action  of  light.  The 
cells  of  the  tissues  will  be  found  unstained. 

Silver  nitrate  is  generally  employed  in  solution  in  the 
following  manner  : — A  1  per  cent,  solution  is  taken,  to  which 
two,  three,  or  four  volumes  of  water  are  added .  The  mode 
of  employment  varies  in  its  details  according  to  circum- 
stances, a  point  which  is  very  important  to  observe.  In  the 
case  of  a  membrane  such  as  the  epiploon,  the  membrane 
must  be  stretched  like  a  drum-head  over  a  porcelain  dish,* 
and  washed  with  distilled  water,  in  order  to  remove  the 
albuminates  and  white  blood-corpuscles  that  are  found  on 
its  surface ;  it  is  then  washed  with  the  solution  of  silver 
nitrate.  In  order  to  obtain  a  powerful  stain  it  is  necessary 
that  this  part  of  the  operation  be  performed  in  direct  sun- 
light, or  at  least  in  a  very  brilliant  light.  As  soon  as  the 
tissue  has  become  white,  and  has  begun  to  turn  of  a  blackish 
grey,  the  membrane  is  removed,  washed  in  distilled  water, 
and  mounted  on  a  slide  in  some  suitable  examination 
medium. 

If  the  membrane  were  left  in  the  water  the  cells  would 
become  detached,  and  would  not  be  found  in  the  finished 
preparation. 

If  the  membrane  had  not  been  stretched  as  directed  the 
silver  would  be  precipitated  not  only  in  the  intercellular 
spaces,  but  in  all  the  small  folds  of  the  surface,  and  the 
forms  of  the  cells  would  be  disguised.  • 

If  the  membrane  had  not  been  washed  with  distilled  water 
before  impregnation  there  would  have  been  formed  a  deposit 
of  silver  on  every  spot  on  which  a  portion  of  an  albuminate 
was  present,  and  these  deposits  might  easily  be  mistaken  for 

*  The  Hoggans  Histological  Rings  will  be  found  much  more  con- 
venient.  They  are  vulcanite  rings  made  in  pairs,  in  which  one  ring  just 
fits  into  the  other,  so  as  to  clip  and  stretch  pieces  of  membrane  between 
them.  They  will  be  found  described  and  figured  in  Journ,  Roy.  Mic.  Soc., 
ii,  1879,  p.  357,  and  in  KOBIN'S  Journ.  de  VAnat.,  1879,  p.  54  They  may 
be  obtained,  in  sets  of  various  sizes  (that  of  seven  eighths  of  an  inch  being 
the  most  convenient  for  3x1  slides),  of  Burge  &  Warren,  42,  Kirby 
Street,  Hatton  Garden,  London,  E.G.,  price  ten  shillings  the  dozen  pairs. 

This  useful  little  apparatus  has  been  re-invented  by  ETERNOD  (Zeit.  f. 
wiss.  Mik.,  iv,  1,  1887,  p.  39),  and  is  made  according  to  his  designs  by 
Demaurex,  Bandagiste,  Fusterie,  Geneva  (Switzerland). 


METALLIC    STAINS    (IMPREGNATION    METHODS).         245 

a  normal  structure  of  the  tissue.  It  is  thus  that  very  often 
impurities  in  the  specimen  have  been  described  as  stomata  of 
the  tissue. 

If  the  solution  be  taken  too  weak — for  instance,  1  :  500  or 
1  :  1000,  or  if  the  light  be  not  brilliant — a  general  instead  of 
an  interstitial  stain  will  result ;  nuclei  will  be  most  stained, 
then  protoplasm,  and  the  intercellular  substance  will  contain 
but  very  little  silver. 

The  tissues  should  be  constantly  agitated  in  the  silver- 
bath  in  order  to  avoid  the  formation  on  their  surfaces  of 
deposits  of  chlorides  and  albuminates  of  silver,  which  would 
give  rise  to  deceptive  appearances. 

In  general  in  a  good  "  impregnation "  the  contents  of 
cells,  and  especially  nuclei,  are  quite  invisible. 

Impregnation  with  silver  may  be  followed  by  treatment 
with  pic  no-carmine  (or  other  carmine  stain),  which  will  bring 
out  the  nuclei,  provided  the  impregnation  has  not  been 
overdone. 

It  should  be  noticed  that  these  impregnations  only  succeed 
with  fresh  tissues,  and  cannot  be  made  to  succeed  with 
tissues  preserved  in  any  way. 


339.  Silver  Nitrate:  the  Solutions  to  be  employed  '(RANVIER). 
— The  solutions  generally  employed  by  RANVIER  vary  in 
strength  from  1  :  300  to  1  :  500.  Thus  1  :  300  is  used  for  the 
epiploon,  pulmonary  endothelium,  cartilage,  tendon  :  whilst 
a  strength  of  1  :  500  is  employed  for  the  study  of  the  phrenic 
centre,  and  for  that  of  the  epithelium  of  the  intestine.  For 
the  impregnation  of  the  endothelium  of  blood-vessels  (by  in- 
jection) solutions  of  1  :  500  to  1  :  800  are  taken. 

M.  DCVAL  (Precis,  p.  229)  recommends  solutions  of  1,  2, 
or  at  most  3  per  cent. 

v.  RECKLINGHAUSEN  used,  for  the  cornea,  a  strength  of 
from  1  :  400  to  1  :  500  (Die  Lymphgefasse,  etc.,  Berlin,  1862, 

p.  5). 

ROBINSKI  (Arch,  de  Physiol.,  1869,  p.  451)  used  solutions 
varying  between  0*1  and  0'2  per  cent.,  which  he  allowed  to 
act  for  thirty  seconds. 

REICH  (Stizb.  d.  ivien.  Akad.,  1873,  Abth.  3,  April;  Zeit. 
f.  wiss.  Mik.,  i,  p.  397)  takes  solutions  of  from  1  :  600  to 


246  CHAPTER   XVIII. 

1  :  400  for  the  study  of  the  endothelium  of  vessels  by  injec- 
tion. 

EODGET  (Arch.  dePhysiol.,  1873,  p.  603)  employed  solutions 
as  weak  as  1  :  750,  or  even  1  :  1000,  exposing  the  tissues  to 
their  action  several  times  over,  and  washing  them  with,  water 
after  each  bath. 

The  HEETWIGS  take,  for  marine  animals,  a  1  per  cent, 
solution  (Jen.  Zeit.  f.  Naturk.,  xvi,  pp.  313  and  324). 

The  HOGGANS  (Journ.  of  Anat.  and  PhysioL,  xv,  1881,. 
p.  477)  take,  for  lymphatics,  a  1  per  cent,  solution. 

TOURNKUX  and  HKRKMANN  (ROBIN'S  Journal  de  V  Anat.,  1876, 
p.  200)  for  the  epithelia  of  Invertebrates  employed  a  solu- 
tion of  3  :  1000  strength,  and  in  some  cases  weaker  solutions. 
The  tissues  were  allowed  to  remain  in  the  silver-bath  for  one 
hour,  and  were  washed  out  with  alcohol  of  90  per  cent. 

HOYER  (Arch.  f.  mik.  Anat.,  1876,  p.  649)  takes  a  solution 
of  nitrate  of  silver  of  known  strength,  and  adds  ammonia  to 
it  until  the  precipitate  that  is  formed  just  redissolves,  then 
dilutes  the  solution  until  it  contains  from  0'75  to  0'50  per 
cent,  of  the  salt. 

This  ammonio-nitrate  solution  is  intended  principally  for 
the  impregnation  of  the  endothelium  of  vessels  by  injection, 
but  can  also  be  used  for  the  impregnation  of  membranes  by 
pouring  on.  It  has  the  advantage  of  impregnating  absolutely 
nothing  but  endothelium  or  epithelium ;  connective  tissue  is 
not  affected  by  it.  It  is  also  said  to  give  a  sharper  localisa- 
tion of  the  stain  than  the  ordinary  solutions. 

DKKHUYSEN  (Anat.  An?..,  iv,  1889,  No.  25,  p.  789;  Zeit.  f. 
iciss.  Mik.,  vii,  3,  1890,  p.  351)  has  applied  to  tissues  of 
terrestrial  animals  the  method  of  HAKMKK  for  marine  animals 
(see  below,  §  343).  For  details  see  previous  editions. 

REGAOD  (Journ.  Anat.  et  Phys.,  xxx.  1894,  p.  719  ;  Zeit.  f. 
u-'iss.  Mik.,  xii,  1,  1895,  p.  74)  recommends  for  the  study  of 
lymphatics  a  process  devised  by  RENAUT,  for  the  details  of 
which  see  also  previous  editions. 

340.  Other  Salts  of  Silver. — ALFEROW  (DuvAL,  Precis,  p.  230)  recom- 
mends the  picrate,  lactate,  acetate,  and  citrate,  as  giving  better  results  than 
the  nitrate.  He  employs  them  in  solutions  of  1 : 800,  and  adds  to  the 
solution  employed  for  staining  a  small  quantity  of  the  acid  of  the  salt 
taken  (10  to  15  drops  of  a  concentrated  solution  of  the  acid  to  800  c.c.  of 
'the  solution  of  the  salt).  The  object  of  the  free  acid  is  to  decompose  the 


METALLIC    STAINS    (IMPREGNATION    METHODS).          247 

precipitates  formed  by  the  action  of  the  silver  salt  on  the  chlorides,  carbo- 
nates, and  other  substances  existing  in  the  tissues. 

341.  Silver  Nitrate  :  Reduction. — Reduction  may  be  effected 
in  other  media  than  distilled  water. 

v.  RECKLINGHAUSKN  washed  his  preparations  in  salt  solution 
before  exposing  them  to  the  light  in  distilled  water  (Arch.  f. 
path.  Anat.,  xix,  p.  45J).  Physiological  salt  solution  (0'75 
per  cent.)  is  commonly  used  for  these  washings. 

MULLER  (Arch.  f.  path.  Anat.,  xxxi,  p.  110),  after  impreg- 
nation by  immersion  for  two  or  three  minutes  in  a  1  per  cent, 
solution  of  nitrate  of  silver. in  the  dark,  adds  to  the  solution 
a  small  quantity  of  1  per  cent,  solution  of  iodide  of  silver 
(dissolved  by  the  aid  of  a  little  iodide  of  potassium).  After 
being  agitated  in  this  mixture  the  preparations  are  washed 
with  distilled  water,  and  exposed  to  the  light  for  two  days 
in  a  1  per  cent,  solution  of  nitrate  of  silver  (see  also  GIERKE, 
in  Zeit.f.  wi**.  Mi'k.,  i,  1884,  p.  396). 

ROUGKT  (Arch.  <\f  Physiol.,  1873,  p.  603)  reduces  in  glyce- 
rin. 

SATTLER  (Arch.  f.  mik.  Anat.,  xxi,  p.  672)  exposes  to.  the 
light  for  a  few  minutes  in  water  acidulated  with  acetic  or 
formic  acid.  THANHOFFER  (Da?  Mikroskop,  1880)  employs  a 
'2  per  cent,  solution  of  acetic  acid. 

KKAUSS  brings  his  preparations,  after  washings,  into  a  light 
red  solution  of  permanganate  of  potash.  Reduction  takes 
place  very  quickly,  even  in  the  dark.  The  method  does  not 
always  succeed  (see  GJEKKE,  in  Zeit.  f.  wiss.  Mik.,  i,  1884r 
]>.  400). 

(  MTITZ  brings  his  preparations  for  two  or  three  minutes 
into  a  0'25  or  0'50  per  cent,  solution  of  chloride  of  tin. 
Reduction  takes  place  very  rapidly  (GiERKhi,  loc.  cit.). 

JAKIMOVITCH  (Jonrn.  ch  I' Anat.,  xxiii,  1888,  p.  142  ;  Journ. 
//"//.  Mic.  Soc.,  1889,  p.  297)  brings  nerve  preparations,  as 
s.»oii  as  they  have  become  of  a  dark  brown  colour,  into  a 
mixture  of  formic  acid  1  part,  amyl  alcohol  1  part,  and  water 
100  parts.  The  objects  exposed  to  the  light  in  this  mixture 
for  two  or  three  days  at  first  become  brighter,  a  part  of  the 
reduced  silver  being  dissolved  :  hence  the  mixture  must  be 
renewed  from  time  to  time.  When  all  the  silver  has  dissolved, 
a  darker  colour  is  permanently  assumed.  The  nerve-cells 
are  left  in  this  mixture  for  five  to  seven  davs. 


248  CHAPTER  XVIII. 

GEEOTA  (Arch.  f.  Anat.  u.  Phys.,  Phys.  AUh.,  1897, 
p.  428;  Zeit.  f.  iviss.  Mik.,  xv,  3,  1899,  p.  348)  greatly 
recommends  reduction  in  a  hydroquinone  developing  solu- 
tion, followed  by  fixation  in  hyposulphite  of  soda,  just  as  in 
photography. 

342.  After-blackening. — LEGEOS     (Journ.  de   VAnat.,  1868, 
p.  275)  washes  his  preparations  after  reduction  in  hyposulphite 
of  soda,  which  he  says  prevents  after-blackening.      Accord- 
ing to  DUVAL   (Precis,  p.  230)  they  should  be  Avashed  for  a 
few  seconds  only  in  2  per  cent,  solution,  and  then  in  dis- 
tilled water. 

343.  Silver  Impregnation  of  Marine  Animals. — On  account 
of  the  considerable  quantity  of  chlorides  that  bathe  the  tissues 
of  marine    animals,    these    cannot   be  treated   directly   with 
nitrate  of  silver. 

HEETWIG  (Jen.  Zeit.,  xiv,  1880,  p.  322)  recommends  fixing 
them  with  a  weak  solution  of  osmic  acid,  then  washing  with 
distilled  water  until  the  wash-water  gives  no  more  than  an 
insignificant  precipitate  with  silver  nitrate,  and  then  treat- 
ing for  six  minutes  with  1  per  cent,  solution  of  silver 
nitrate. 

HAKMEE  (Mitth.  Zool.  Stat.  Neapel,  v,  1884,  p.  445) 
washes  them  for  some  time  (half  an  hour)  in  a  5  per  cent, 
solution  of  nitrate  of  potash  in  distilled  water  ;  they  may 
then  be  treated  with  silver  nitrate  in  the  usual  way.  This 
method  gave  good  results  Avith  Loxosoma  and  Pedicellinu, 
with  Medusae,  Hydroids,  Sagitta  and  Appendicularia. 

HAEMEE  thinks  that  for  some  animals  other  solutions 
having  the  same  density  as  sea  water  might  be  substituted 
for  the  nitrate  of  potash,  and  recommends  a  4'5  per  cent, 
solution  of  sulphate  of  soda. 

344.  Impregnation  of  Nerve  Tissue. — For  this  subject,  which 
includes    the    important    bichromate-and-silver    method    of 
GOLGI,  see  Part  II. 

345.  Double -staining    Silver-stained  Tissues. — The  nuclei  of 
tissues    impregnated   with    silver   may  be   stained  with   the 
usual  reagents,  provided  that  solutions   containing  free   am- 


METALLIC    STAINS    (IMPREGNATION    METHODS).          249 

monia  be  avoided,  as  this  would  dissolve  out  the  silver. 
These  stains  will  only  succeed,  however,  with  successful 
negative  impregnations,  as  nuclei  that  have  been  impreg- 
nated will  not  take  the  second  stain. 

Impregnation  with  silver  may  be  followed  by  impregna- 
tion with  gold.  In  this  case  the  gold  generally  substitutes 
itself  for  the  silver  in  the  tissues,  and  though  the  results  are 
sharp  and  precise,  the  effect  of  a  double  stain  is  not  pro- 
duced. See  hereoii  GEROTA,  loc.  cit.  §  341. 


Gold. 

346.  The  Characters  of  Gold  Impregnations. — Gold  chloride 
differs  from  nitrate  of  silver  in  that  it  generally  gives  positive 
(§  334)  impregnations  only.  It  only  gives  negative  images, 
so  far  as  I  know,  when  caused  to  act  on  tissues  that  have 
first  received  a  negative  impregnation  with  silver,  the  gold 
substituting  itself  for  the  silver.  In  order  to  obtain  these 
images  you  first  impregnate  very  lightly  with  silver  ;  reduce  ; 
treat  for  a  few  minutes  with  a  0*5  per  cent,  solution  of  gold 
chloride,  and  reduce  in  acidulated  distilled  water. 

This  process,  however,  is  in  but  little  use,  and  except  for 
the  staining  of  cytoplasm  for  cytological  researches  and  for 
certain  special  studies  on  the  cornea,  and  on  connective 
tissue,  the  almost  exclusive  function  of  gold  chloride  is  the 
impregnation  of  nervous  tissue.  For  this  tissue,  gold  chloride 
exhibits  a  remarkable  selectivity,  in  virtue  of  which  it  justly 
ranks  as  a  most  valuable  reagent  for  the  study  of  nerve  end- 
organs  and  the  distribution  of  nerves. 

For  all  the  objects  above  named  gold  chloride  is  capable 
of  furnishing  preparations  that  for  beauty  and  clearness 
cannot  be  surpassed,  if  even  they  can  be  equalled  by  any 
other  means.  But  not  every  gold  preparation  is  successful. 
Further,  gold  chloride  is  very  uncertain  in  its  action. 

It  is  now  acknowledged  that  the  very  best  gold  preparations 
give  images  that  are  only  worthy  of  credence  as  to  what 
they  show,  and  furnish  absolutely  no1  evidence  whatever  as 
to  the  non-existence  of  anything  that  they  do  not  show ;  for 
you  can  never  be  sure  that  the  imbibition  of  the  salt  has 
not  capriciously  failed,  or  its  reduction  capriciously  stopped 


250  CHAPTER   XVIII. 

at  any  point.  Thafc  the  images  frequently  do  stop  caprici- 
ously short  in  the  representation  of  reality  there  is  abundant 
evidence. 

Few  of  the  methods  about  to  be  described  give  perfectly 
permanent  preparations.  Most  of  them  will  not  retain  all 
their  beauty  for  more  than  a  few  weeks.  Still,  the  greater 
the  care  taken  in  preparation,  and  particularly  the  greater 
the  care  taken  to  ensure  thorough  reduction  of  the  gold,  the 
longer  will  be  the  life  of  the  preparations. 

Careful  attention  to  the  devices  to  this  end  detailed  in 
the  following  paragraphs  will  do  much  ;  and  possibly  LINDSAY 
JOHNSON'S  suggestion  (supra,  §  336)  of  the  utility  of  "  sun- 
ning "  the  solutions  before  use  may  prove  an  unexpected 
help. 

347.  As  to  the  Commercial  Salts  of  Gold. — SQUIRE'S  Method* 
<uid    Formulte,   etc.   (p.   43),  an    excellent    authority  on  the 
chemistry  of  histological  reagents,  says :  "  Commercial  chloride 
of  gold  is  not  the  pure  chloride,  AuCl3,  but   the  crystallised 
double  chloride  of  gold  and  sodium,  containing  50  per  cent, 
of  metallic  gold. 

"  Commercial  chloride  of  gold  and  sodium  is  the  above 
crystallised  double  chloride  mixed  with  an  equal  weight  of 
chloride  of  sodium,  and  contains  25  per  cent,  of  metallic 
gold." 

This,  however,  appears  not  to  be  the  case  in  Germany. 
Dr.  GRUBLEU,  writing  to  MAYER  (seethe  Grundziic/e,  LEE  und 
MAYER,  p.  215)  says  :  "  Aurtim  chloratum  fusaum  contains 
about  53  per  cent.  Au,  the  fiaonm  about  48  per  cent. ;  in 
both  of  them  there  should  be  only  water  and  hydrochloric 
acid  besides  the  gold,  no  sodium  chloride.  Pure  Auronatrium 
chloratum  contains  14' 7  per  cent,  of  sodium  chloride,  though 
samples  are  found  in  commerce  with  much  more." 

APATHY  (Mitth.  ZooL  Stat.  Neapel,  xii,  1897,  p.  722) 
formerly  employed  the  aiirum  chloratum  Jtavum,  but  now 
prefers  the  fuscunt. 

348.  Foregilding  and  Aftergilding.— Gold  methods  may  be 
divided  into  two  groups  ;  the  one,  chiefly  concerned  with  the 
study  of  peripheral  nerves  or  nerve   end-organs,  is  charac- 
terised by  employing  either  perfectly  fresh  tissues  or  tissues- 


METALLIC    STAIJNS    (iMPKKGNATION    METHODS).         251 

that  have  been  subjected  to  a  special  treatment  by  organic 
acids  ;  the  other,  concerned  chiefly  with  the  study  of  nerve- 
centretj  is  characterised  by  the  employment  of  fixed  and 
hardened  tissues. 

These  two  groups  of  methods  may  be  distinguished  with 
APATHY  as  the  Foregilding  methods  (Vorcergoldung)  t  and.  the 
After  gilding  methods  (Nachvergoldung).  Amongst  the  latter 
is  one  at  least,  that  of  APATHY,  which  affords  not  only  a  stain 
of  nervous  tissue,  but  also  an  excellent  nuclear  and  plasmatic 
stain  of  tissues  in  general. 


A.  Foregilding. 

349.  The  State  of  the  Tissues  to  be  impregnated. — The  once 
classical  rule,  that  for  researches  on  nerve-endings  the  tissues 
should  be  taken  perfectly  fresh,  seems  not  to  be  valid  for  all 
cases.      For  DKASCB   (Sitzb.   k.  k.  Akad.  Wins.    Wien,   1881, 
p.  171,  and  1884,  p.  510;  and  Abhand.  math.-phys.  CL  d.  K. 
>''/>•/,.  (lex.  <!.  ir/W.,  xiv,  No.  5,  1887;  Zeit.  f.  wiss.  Mik.,  ivr 
4,  1887,  p.  492)  finds  that  better  results  are  obtained  with 
tissues  that  have  been  allowed  to  lie  after  death  for  twelve, 
twenty-four,  or  even  forty-eight  hours  in  a  cool  place.      He 
even  suspects  that   the  function  of  the  organic  acids  in  the 
methods  inspired  by  Lowi'i's  method  is  to  bring  the  tissues 
into  somewhat  the  state  in  which  they  are  naturally  found  at 
a  certain  moment  of  post-mortem  process — a  state,  namely, 
in  which  the  nerves  have  a  special  susceptibility  for  impreg- 
nation with  gold. 

350.  COHNHEIM'S  Method    (Vurchow**  Arch.,   Bd.    xxxviii, 
pp.  346—349;  Strick'-r's  Handb.,  p.  1100).— This,  the  arche- 
type of  the  gold  methods,  was  as  follows  : — Fresh  pieces  of 
cornea  (or  other  tissue)  are  put  into  solution  of  chloride  of 
gold  of   0'5  per   cent,  strength    until   they  are   thoroughly 
yellow,  and  then  exposed  to  the   light   in   water   acidulated 
with  acetic  acid  until  the  gold  is  thoroughly  reduced,  which 
happens  in  the  course  of  a  few  days  at  latest.      They  are  then 
mounted  in  acidulated  glycerin. 

The  method  in  this,  its  primitive  form,  often  gave  splendid 
results,  but  was  very  uncertain,  giving  sometimes  a   nuclear 


252  CHAPTER   XVIII. 

or  protoplasmic  stain,  sometimes  an  extra- cellular  impregna- 
tion similar  to  that  of  nitrate  of  silver.  And  the  preparations 
thus  obtained  are  anything  but  permanent. 

351.  LOWIT'S  Method   (Sitzgsber.  Akad.    Wien,    Bd.    Ixxi, 
Abth.  3,  1875,  p.  1). — The  principle  of  this  process  is  that, 
in  order  to  facilitate  the  penetration  of  the  gold  and  its  sub- 
sequent   reduction  in  the  tissues,  the   tissues    are    made    to 
swell  up  by  treatment  with  formic  acid  before  being  brought 
into  the  gold-bath,  and  formic  acid  is  employed  to  assist  the 
reduction  after  impregnation. 

The  following  directions,  which  may  serve  as  a  type,  are 
ta.ken  from  FISCHER'S  paper  on  the  corpuscles  of  Meissner 
(Arch.f.  mik.  Anat.,xii,  1875,  p.  366). 

Small  pieces  of  fresh  skin  are  put  into  dilute  formic  acid 
(one  volume  of  water  to  one  of  the  acid  of  1*12  sp.  gr.),aiid 
remain  there  until  the  epidermis  peels  off.  They  then  are 
put  for  fifteen  minutes  into  gold  chloride  solution  (1£  per 
cent,  to  1  per  cent.),  then  for  twenty-four  hours  into  dilute 
formic  acid  (1  part  of  the  acid  to  1 — 3  of  water),  and  then 
for  twenty-four  hours  into  undiluted  formic  acid.  (Both  of 
these  stages  are  gone  through  in  the  dark.)  Thin  sections 
are  then  made  and  mounted  in  dammar  or  glycerin.  Suc- 
cessful preparations  show  the  nerves  alone  stained,  but  it  is 
not  possible  always  to  control  the  results. 

352.  RANviuii's  Formic  Acid  Method  (Quart,  Journ.  Hie.  Sci. 
[N.  S.J,  Ixxx,  1880,  p.  456).— The  method  of  LOWIT  has  been 
modified  by  many  workers  by   omitting  the   final  treatment 
with  undiluted  formic  acid,  and  also  in  some   other  details. 
RAN vi KB  proceeds  as  follows  : — Reflecting  that  the  action  of 
the  one  third  formic  acid  in  which  LOWIT  placed  his  tissues 
must  be  hurtful  to  the  final  ramifications  of  the  nerves,  he 
combines  the  formic    acid  with  a  fixing    agent   designed  to 
antagonise  its  altering  action,  and  takes  for  this  purpose  the 
chloride  of  gold  itself.      The  tissues  are  placed  in  a  mixture 
of  chloride  of  gold  and  formic  acid  (four  parts  of  1  per  cent, 
gold   chloride  to    one  part  of   formic  acid)   which   has  been 
boiled  and  allowed  to  cool  (RANVIER'S  Trait/',  p.  826).      They 
remain  in  this  until  thoroughly  impregnated   (muscle  twenty 
minutes,  epidermis  two  to  four  hours)  ;  the  reduction  of  the 


METALLIC    STAINS    (IMPREGNATION    METHODS).         253 

gold  is  effected  either  by  the  action  of  daylight  in  acidulated 
water,  or  in  the  dark  in  dilute  formic  acid  (one  part  of  the 
acid  to  four  parts  of  water). 

The  object  of  boiling  the  mixture  of  gold  chloride  and 
formic  acid  is  this,  that  "  by  boiling  in  the  presence  of  the 
acid  the  gold  acquires  a  great  tendency  to  reduction,  and 
for  this  reason  its  selective  action  on  nervous  tissues  is 
enhanced/' 


353.  KANVIER'S  Lemon-juice  Method  (Trait<\  p.  831).— 
RAXVIER  finds  that  of  all  acids  lemon  juice  is  the  least  hurtful 
to  nerve-endings.  He  therefore  soaks  pieces  of  tissue  in 
fresh  lemon  juice,  filtered  through  flannel,  until  they  become 
transparent  (five  or  ten  minutes  in  the  case  of  muscle). 
They  are  then  rapidly  washed  in  water,  brought  for  about 
twenty  minutes  into  1  per  cent,  gold  chloride  solution, 
washed  again  in  water,  and  brought  into  a  bottle  containing 
50  c.c.  of  distilled  water  and  two  drops  of  acetic  acid.  They 
are  exposed  to  the  light  for  twenty-four  to  forty-eight  hours. 
The  preparations  thus  obtained  are  good  for  immediate  study,, 
but  are  not  permanent,  the  reduction  of  the  gold  being  in- 
complete. In  order  to  obtain  perfectly  reduced,  and  there- 
fore permanent,  preparations,  the  reduction  should  be  done 
in  the  dark  in  a  few  cubic  centimetres  of  dilu  e  formic  acid 
(1  part  acid  to  4  of  water).  The  reduction  is  complete  in 
twenty-four  hours. 

354.  VIALLANE'S    Osmic   Acid  Method     (Hist,   et  Dev.    des 
Insectes,  1883,  p.  42). — The  tissues  are  treated   with   osmic 
acid   (1   per  cent,  solution)   until  they  begin  to  turn  brown, 
then  with   25   per  cent,  formic  acid  for  ten  minutes;   they 
are  then  put  into  solution  of  chloride  of  gold  of  1  :  5000  (or 
even  much  weaker)  for  twenty-four  hours  in  the  dark,  then 
reduced  in  the  light  in  25  per  cent,  formic  acid.      According 
to  my  experience  this  is  an  excellent  method. 

355.  Other  Methods. — The   numerous   other  methods   that 
have    been    proposed    differ  from   the    foregoing   partly   in 
respect  of  the   solutions  used  for  impregnation,  but  chiefly 
in  respect  of  details  imagined  for  the  purpose  of  facilitating 


254  CHAPTER   XVI1T. 

the   reduction  of  the  gold,,  and  rendering  it  as  complete  as 
possible. 

Thus  BASTIAN  modified  COHNH KIM'S  original  method  by 
employing  a  solution  of  gold  chloride  of  a  strength  of  1  to 
2000,,  acidulated  with  HC1  (1  drop  to  75  c.c.),  and  perform- 
ing the  reduction  in  a  mixture  of  equal  parts  of  formic  acid 
:and  water  kept  warm,  heat  being  an  agent  that  furthers 
reduction. 

HENOCQUK  (Arch,  dc  I'Anat.  et  de  la  PhysioL,  1870,  p.  Ill) 
impregnates  in  a  0'5  per  cent,  solution  of  gold  chloride,, 
washes  in  water  for  twelve  to  twenty-four  hours,,  and  re- 
duces,, with  the  aid  of  heat,,  in  a  nearly  saturated  solution  of 
tartaric  acid.  The  tartaric  acid  solution  must  be  contained 
in  a  well-stoppered  bottle.  The  best  temperature  for  reduc- 
tion is  40°  to  50°  C.  Reduction  is  effected  very  rapidly, 
.sometimes  in  a  quarter  of  an  hour. 

This  process  has  been  described  as  the  method  of  CHRSCHT- 
•suHONOWtc  (Arch.  f.  mile.  Anat.,  vii,  1872,  p.  383). 

HOYER  (Arch.  f.  mik.  Anat.,  ix,  1873,  p.  222)  says  that 
the  double  chloride  of  gold  and  potassium  has  the  following- 
advantages  over  the  simple  gold  chloride.  It  is  more  easy 
to  be  obtained  of  unvarying  composition,  it  is  more  per- 
fectly neutral,  and  its  solutions  are  more  perfectly  stable. 
He  uses  it  in  solutions  of  the  same  strength  as  chloride  of 
gold,  viz.  0'5  per  cent.  In  order  to  demonstrate  the  intra- 
•epithelial  ramifications  of  nerves  of  the  cornea,  the  gold  is 
partially  reduced  by  exposure  of  the  tissue,  after  impregna- 
tion for  sixteen  to  twenty-four  hours  in  (one  or  two  ounces 
of)  distilled  water,  and  there  are  added  to  the  water  one  or 
two  drops  of  a  pyrogallic  acid  developing  solution,  such  as 
is  used  in  photography  (vide  G-EKLACH,  Die  Photographic  als 
Hiilfsmittel  der  mikrosltopischen  Forsvhnng,  Leipzig,  1863). 
Or  instead  of  treating  them  with  the  developing  solution,  the 
cornea  may  be  removed  to  a  warm  concentrated  solution  of 
tartaric  acid,  and  remain  there  at  the  temperature  of  an  in- 
cubating stove  until  the  gold  is  fully  reduced. 

I  have  myself  used  the  double  chloride  of  gold  and  sodium 
with  good  results. 

CIACCIO  (Joimi.  de  Microgr.,  vii,  1883,  p.  38;  Journ.  Boy. 
Mic.  Soc.  [N.S.],iii,  1883,  p.  290)  prefers  the  double  chloride 
of  gold  and  cadmium. 


METALLIC   STAINS    (IMPREGNATION  METHODS).          255 

FLECHSIG  (Die  Leitungsbahnen  in  Gehirn,  1876  ;  Arch.  f. 
Anat.  u.  Phys.,  1884,  p.  453)  reduces  in  a  10  per  cent,  solu- 
tion of  caustic  soda. 

NESTEKOFFSKY  treats  impregnated  preparations  with  a  drop 
•of  sulphydrate  of  ammonium,  and  finishes  the  reduction  in 
glycerin  (quoted  from  G-IEKKE'S  Farberei  z.  mik.  Zwecken). 

BOHM  reduces  in  PRITC HARD'S  solution — amyl  alcohol ,  1  ; 
formic  acid,  1 ;  water,  98. 

MANFRED i  treats  fresh  tissues  as  follows  (Arch,  per  le  Set. 
med.,  v,  No.  15)  : — Gold  chloride,  1  per  cent.,  half  an  hour ; 
•oxalic  acid,  0*5  per  cent.,  in  which  they  are  warmed  in  a 
water-bath  to  36°,  allowed  to  cool,  and  examined.  Mount 
in  glycerin.  Sunny  weather  is  necessary. 

BOCCARDI  (Lavori  Ins  tit.  Fisiol.  Napoli,  1886,  i,  p.  27  ; 
Journ.  Roy.  Hie.  Soc.,  1888,  p.  155)  recommends  oxalic  acid 
•of  0*1  per  cent,  or  of  0'25  to  0'3  per  cent.,  or  a  mixture  of 
5  c.c.  pure  formic  acid,  1  c.c.  of  1  per  cent,  oxalic  acid,  and 
25  c.c.  of  water.  Objects  should  remain  in  this  fluid  in  the 
dark  not  longer  than  two  to  four  hours. 

KOLOSSOW  (Zeit.f.  winy.  Mik.,  v,  1,  1888,  p.  52)  impregnates 
for  two  or  three  hours  in  a  1  per  cent,  solution  of  gold  chloride 
acidulated  with  1  per  cent,  of  HC1,  and  reduces  for  two  or 
three  days  in  the  dark  in  a  0*01  per  cent,  to  0*02  per  cent, 
solution  of  chromic  acid. 

GKBERG  (Intern.  Monatxschr.,  x,  1893,  p.  205)  states  that 
previous  treatment  of  tissues  for  twenty- four  hours  with  lime- 
water  (AKNSTEIN'S  method)  greatly  helps  the  reduction. 

BKRNHEIM  (Arch.  f.  Anat.  u.  Phys.,  Phys.  Abth.,  1892, 
Supp.,  p.  29  ;  Zeit.  f.  wiss.  Mik,,  x,  4,  1893,  p.  484)  adds  to 
Lo WIT'S  dilute  formic  acid  a  piece  of  sulphite  of  sodium 
(must  be  fresh  and  smell  strongly  of  sulphurous  acid). 

Dr.  LINDSAY  JOHNSON  writes  to  me  that  besides  the  "  sun- 
ning" of  the  impregnating  solution  recommended  above 
(§  336),  the  following  precautions  should  be  taken: — "  The 
tissue  must  be  well  washed  in  distilled  water,  and  the  gold 
•carefully  acidulated  with  a  neutral  acetate  or  formiate,  or 
acetic  or  formic  acid,  at  least  twenty-four  hours  before  using  ; 
and  then  afterwards  the  tissue  must  be  washed  until  no  re- 
action occurs  to  test-paper. " 

APATHY  (Mikrotechnik,  p.  173 ;  Mitth.  Zool.  Stat.  Neapel, 
xii,  1897,  pp.  718 — 728)  lays  stress  on  the  necessity  of 


256  CHAPTER    XVIII. 

having  the  objects  thoroughly  penetrated  by  light  from  all 
sides  during  the  process  of  reduction.  Objects,  therefore, 
should  always  be  so  thin  that  light  can  readily  stream 
through  them  ;  they  should  either  be  membranes  or  sections. 
They  should  be  either  stretched  out  (e.g.  on  a  slide)  or  hung 
up  in  the  reducing  bath  in  such  a  way  as  to  be  lighted  from 
both  sides.  He  impregnates  for  a  few  hours  in  1  per  cent, 
gold  chloride  (§  347)  in  the  dark,  then  brings  the  objects 
Avithout  washing  out  with  water,  the  gold  solution  being 
just  superficially  mopped  up  with  blotting-paper,  into  1  per 
cent,  formic  acid.  They  are  to  be  set  up  in  this,  in  a  tube 
or  otherwise,  so  that  the  light  may  come  through  them  from 
all  sides,  and  exposed  to  diffused  daylight  in  summer,  or 
direct  sunlight  in  winter,  for  six  to  eight  hours  without  a 
break.  They  must  not  be  moved  about  more  than  can  be 
helped  in  the  acid.  If  the  acid  becomes  brown  it  may  be 
changed  for  fresh.  The  temperature  of  the  acid  should  not 
be  allowed  to  rise  over  20°  C.,  whence  direct  sunlight  is  to 
be  avoided  during  the  summer.  He  mounts  in  glycerin 
or  his  syrup  (§  328) .  He  finds  such  preparations  absolutely 
permanent. 

B.  After  gilding . 

356.  GEBLACH'S  Method  (STRIKER'S  Handb.,  1872,  p.  678)  : 
— Spinal  cord  is  hardened  for  fifteen  to    twenty   days  in  a 
1  to  2  per  cent,  solution  of  bichromate   of  ammonia.      Thin 
sections  are  made  and  thrown  into  a  solution  of   1   part  of 
double  chloride  of  gold  and  potassium  to  10,000  parts  water, 
which  is  very  slightly  acidulated  with   HC1.      They   remain 
there  from  ten  to  twelve  hours,  and  having  become  slightly 
violet,    are    washed   in   hydrochloric    acid    of    1    to   2  : 3000 
strength,  then  brought  for  ten  minutes  into  a  mixture  of   1 
part  HC1  to  1000  parts  of  60  per  cent,  alcohol,  then  for  a  few 
minutes  into  absolute  alcohol,  and  thence  into  clove  oil,  for 
mounting  in  balsam. 

(See  further,   for    Nerve   Centres,   under    fi  Neurological 
Methods.") 

357.  GOLGI  (Mem.  Accad.  Torino  [2],  xxxii,  1880,  p.  382) 
treats  tissues  previously  hardened  in  2  per  cent,  solution  of 


METALLIC    STAINS    (IMPREGNATION  METHODS).          257 

bichromate  of  potash,  as  follows  : — They  are  put  for  ten  to 
twenty  minutes  into  1  per  cent,  solution  of  arsenic  acid,  then 
into  J  per  cent,  solution  of  chloride  of  gold  and  potassium 
for  half  an  hour,  washed  in  water,  and  reduced  in  sunlight 
in  1  per  cent,  arsenic  acid  solution,  which  is  changed  for 
fresh  as  fast  as  it  becomes  brown.  Mount  in  glycerin. 
Sunny  weather  is  necessary. 

358.  APATHY'S  Method  (Zeit.  f.  wiss.  Mik.,  x,  1893,  p.  349  ; 
Mitth.  Zool.  Stat.  Neapel,  xii,  1897,  p.  729 ;  Zeit.f.  wiss.  Mik., 
xv,  1,  1898,  p.  79). — The  material  to  be  used  must  have 
been  fixed  either  in  sublimate  or  in  a  mixture  of  equal 
parts  of  saturated  solution  of  sublimate  in  0*5  per  cent,  salt 
solution  and  1  per  cent,  osmic  acid  (this  more  particularly 
for  Vertebrates).  The  material  should  be  imbedded  as 
quickly  as  possible,  either  in  paraffin  or  in  celloidin.  The 
paraffin  material  will  keep  in  a  good  state  indefinitely,  and 
so  will  the  celloidin  material  provided  that  the  blocks  be 
preserved  in  a  thick  solution  of  glycerin  jelly  with  a  piece 
of  thymol  in  it  (the  jelly  is  removed  before  cutting  by 
warming  and  washing  with  warm  water).  Sections  are 
made  when  desired  and  fixed  on  slides,  and  after  the  usual 
treatment  with  iodine,  etc.,  are  either  put  into  distilled 
water  for  from  two  to  six  hours,  or  are  rinsed  in  water, 
treated  for  one  minute  with  1  per  cent,  formic  acid,  and 
again  well  washed  with  water. 

They  are  then  put  for  twenty-four  hours,  or  at  least  over- 
night, into  the  gold-bath,  which  is  preferably  1  per  cent, 
gold  chloride  (see  §  347),  but  may  be  weaker,  down  to  O'l 
per  cent.,  after  which  they  are  just  rinsed  with  water  or 
superficially  dried  with  blotting-paper.  The  slides  are  then 
set  up  on  end  in  a  sloping  position,  the  sections  looking 
downwards,  so  that  precipitates  may  not  fall  on  them,  in  glass 
tubes  filled  with  1  per  cent,  formic  acid.  The  tubes  are 
then  exposed  to  light  until  the  gold  is  reduced,  as  directed 
in  §  355  (you  may  set  them  up  near  a  window,  and  place  a 
reflector  of  some  sort  behind  them  ;  a  sheet  of  white  paper 
will  do).  After  reduction  you  may  counterstain,  if  desired, 
and  mount  in  any  way.  After  trial  I  highly  recommend 
this  process.  I  have  found  it  advantageous  to  reduce  in 
weak  solution  of  formaldehyde,  either  with  or  without 

17 


258  CHAPTER  XVIII. 

formic  acid.      A  few  drops  of  formol  added  to  the  tube  with 
the  objects  will  suffice. 

359,  Impregnation  of  Marine  Animals.  — For    some    reason 
that  I  am  unable  to  explain,  the  tissues  of  marine  animals 
do  not  readily  impregnate  with  gold  in  the   fresh  state.      It 
is  said  by*  FOL  that  impregnation  succeeds  better  with  spirit 
specimens. 

360.  Ulterior  Treatment  of  Impregnated  Preparations. — Pre- 
parations may  be  mounted  either  in  balsam  or  in  acidulated 
glycerin  (1  per  cent,  formic  acid). 

Theoretically  they  ought  to  be  permanent  if  the  reduction 
of  the  metal  has  been  completely  effected,  but  they  are  very 
liable  to  go  wrong  through  after-blackening.  RANVIER  states 
that  this  can  be  avoided  by  putting  the  preparations  for  a 
few  days  into  alcohol,  which  he  says  possesses  the  property 
of  stopping  the  reduction  of  the  gold.  But  this  must  be 
taken  to  mean  that  by  this  device  the  period  of  usefulness 
of  the  preparations  may  be  prolonged  for  some  time,  not 
indefinitely. 

Blackened  preparations  may  be  bleached  with  cyanide  or 
ferrocyanide  of  potassium.  REDDING  employs  a  weak  solution 
of  ferrocyanide  ;  CYBULSKY  a  0*5  per  cent,  solution  of  cyanide. 
But  the  results  are  far  from  being  perfectly  satisfactory. 

Preparations  may  be  double-stained  with  the  usual  stains 
(safranin  being  very  much  to  be  recommended),  but  nuclei 
will  only  take  the  second  stain  in  the  case  of  negative  im- 
pregnation. 


Other  Metallic  St 


ains. 


361.  Osmic  Acid  and  Pyrogallol.— This  method  was  first 
published  by  me  in  1887  (La  Cellule,  t.  iv,  fasc.  1,  p.  110). 
It  consists  essentially  in  putting  tissues  that  have  been 
treated  with  osmic  acid  into  a  weak  solution  of  pyrogallol, 
in  which  they  quickly  turn  greenish  black,  sometimes  much 
too  much  so. 

At  that  time  I  had  only  tried  the  reaction  with  tissues 
impregnated  with  pure  osmic  acid.  Since  then  HERMANN 
(Arch.  f.  mik.  Anat.y  xxxvii,  4,  1891,  p.  570)  tried  it  with 


METALLIC   STAINS   (IMPREGNATION   METHODS).          259 

tissues  fixed  in  his  platino-aceto-osmic  mixture  (§  50).  With 
this  modification  of  the  process  incomparably  better  results 
are  obtained. 

.  HERMANN'S  procedure  is  as  follows  : — The  tissues  are  put 
for  one  or  two  days  into  the  platino-aceto-osmic  mixture, 
washed  thoroughly  in  water,  and  hardened  in  successive 
alcohols ;  after  which,  to  obtain  the  black  reaction,  they  are 
put  for  twelve  to  eighteen  hours  into  raw  pyroligneous  acid. 
This  acid  ought  (Ergebnisse  der  Anat.,  ii,  1893,  p.  28)  to  be 
as  raw  as  possible,  and  to  be  of  a  dark  brown  colour  and 
evil-smelling.  (The  stain  obtained  in  this  way  is  not  due  to 
a  mere  reduction  of  the  osmic  acid,  but  also  to  coloration  by 
the  brown  pyroligneous  acid ;  for  HERMANN  has  obtained  the 
same  stain  with  sublimate  material,  or  alcohol  material  [op. 
cit.,  i,  1891  (1892),  p.  7]). 

According  to  my  experience,  the  procedure  of  HERMANN 
is  not  that  which  gives  the  best  results.  I  now  proceed  as 
follows  : 

Either  the  mixture  of  HERMANN  or  the  mixture  of  FLEMMING 
may  be  used  for  fixing.  You  may  leave  the  tissues  therein 
for  twelve  or  twenty-four  hours  if  you  think  that  this  is 
desirable  in  the  interest  of  a  complete  fixation ;  but  in  the 
interest  of  the  stain  alone,  half  an  hour  is  enough  and  is 
preferable.  It  is  not  only  useless  but  hurtful  to  put  the 
preparations  into  alcohol  after  fixation,  for  it  is  desirable 
that  the  tissue  should  be  in  as  fresh  a  state  as  possible  on 
coming  into  the  pyrogallol.  In  consequence  it  is  not  possible 
to  obtain  the  best  results  by  treating  paraffin  sections.  The 
tissues  are  therefore  brought  in  bulk,  directly  after  fixing, 
into  pyrogallol.  You  may  use  Hermann's  pyroligneous  acid 
if  you  like,  but  I  consider  that  a  weak  solution  of  pyrogallol 
is  preferable.  The  tissues  may  remain  in  either  of  these 
liquids  for  twenty-four  hours,  but  for  small  objects  an  hour 
or  less  is  sufficient.  An  alcoholic  solution  of  pyrogallol  may 
be  taken  if  desired,  and  this  way  be  indicated  in  certain 
cases.  I  have  not  obtained  the  reaction  with  tannin  used  on 
chrom-osmium  material. 

There  is  thus  obtained  a  black  stain,  which  is  at  the  same 
time  a  plasma  stain  and  a  nuclear  stain,  chromatin  being  so 
far  stained  that  it  is  not  necessary  to  have  recourse  after- 
wards to  a  special  chromatin  stain.  This  is  one  of  the  best 


260  CHAPTER   XVIII. 

methods  that  I  know  of  for  the  study  of  Nebenkerne.  With 
Invertebrates  it  sometimes  gives  very  elegant  differentiations 
of  nervous  tissue.  It  is  a  very  easy  method,  and  if  pyrogallol 
be  used  a  very  safe  one  (with  pyroligneous  acid  not  so  safe). 
Although,  as  said,  this  method  enables  one  to  dispense 
with  a  special  second  chromatin  stain,  yet  it  is  frequently 
very  advantageous  to  use  one.  I  greatly  recommend  safranin 
(stain  very  strongly,  twenty-four  hours  at  least,  and  start 
the  extraction  with  acid  alcohol). 

This  method  has  been  attributed  to  VON  MAEHRENTHAL.  A  modification 
of  this  method  is  said  by  AZOULAY  to  give  a  specific  stain  of  the  medullary 
sheath  of  nerves;  see  his  process  under  "  Neurological  Methods  "  in  Part  II. 
See  also  a  similar  process  for  medullated  nerve  by  HELLER  and  GUMPERTZ, 
quoted  Zeit.f.  wiss.  Mik.,  xii,  3,  1896,  p.  385. 

The  communications  of  KOLOSSOW  (Zeit.f.  wiss.  Mik.,  ix,  1,  1892,  p.  38, 
and  ix,  3,  1893,  p.  316)  do  not  appear  to  me  to  constitute  a  useful  con- 
tribution to  the  subject. 

362.  Perchloride  of  Iron. — This  reagent,  introduced  by  POLAILLON 
(Journ.  de  VAnat.,  iii,  1866,  p.  43),  sometimes  gives  useful,  results,  especi- 
ally in  the  study  of  peripheral  nerve-ganglia,  in  which  it  stains  the  nervous 
tissue  alone,  the  connective  tissue  remaining  colourless.     The  method  con- 
sists in  impregnating  in  perchloride  of  iron,  and  reducing  in  tannic,  gallic, 
or  pyrogallic  acid. 

The  HOGGANS  proceed  as  follows  (Journ.  Quekett  Club,  1876;  Journ. 
Roy.  Mic.  Soc.,  ii,  1879,  p.  358) : — The  tissue  (having  been  first  fixed  with 
silver  nitrate,  which  is  somewhat  reduced  by  a  short  exposure  to  diffused 
light)  is  dehydrated  in  alcohol,  and  treated  for  a  few  minutes  with  2  per 
cent,  solution  of  perchloride  of  iron  in  spirit.  It  is  then  treated  with  a 
2  per  cent,  solution  of  pyrogallic  acid  in  spirit,  and  in  a  few  minutes  more, 
according  to  the  depth  of  tint  required,  may  be  washed  in  water  and 
mounted  in  glycerin. 

FOL  (see  ante,  §  80)  fixes  in  perchloride  solution,  and  treats  the  prepara- 
tions for  twenty. four  hours  with  alcohol  containing  a  trace  of  gallic  acid. 

POLAILLON  (loc.  cit.")  reduces  in  tannic  acid. 

This  method  is  not  applicable  to  chromic  objects. 

I  have  found  it  useful  in  certain  special  cases. 

363.  Pyrogallate  of  Iron  (KOOSEVELT,  Med.  Rec.,  ii,  1817,  p.  84 ; 
Journ.  Roy.  Mic.  Soc.,  1888,  p.  157). — A  stain  composed  of  20  drops  of 
saturated  solution  of  iron  sulphate,  30  grms.  water,  and  15  to  20  drops 
pyrogallic  acid. 

364.  Palladium  Chloride  (see  P.  SCHULTZE,  ante,  §  78).     Prussian 
Blue  (see  LEBER,  Arch.  f.   Ophthalm.,  xiv,  p.  300;   EANVIER,  Traite, 
p.  108).     Cupric  Sulphate  (see  LEBER,  ibid.).    Lead  Ohromate  (see 


METALLIC    STAINS    (IMPREGNATION    METHODS).        261 

LEBKE,  ibid.).  Sulphides  (see  LAXDOIS,  Centralb.  f.  d.  tncd.  Wiss., 
1885,  No.  55 ;  and  GIEEKE,  in  Zeit.f.  wiss.  Mile.,  i,  1884,  p.  497).  Molyb- 
date  of  Ammonium  (MEEKEL  ;  KEAUSE)  (see  GIEBKE,  Zeit.f.  wiss.  Mik., 
i,  1884,  p.  96).  Oxychloride  of  Ruthenium  (NicoLLE  and  CANTACUZENE) 
(see  Ann.  List.  Pasteur,  Tii,  1893,  p.  331).  Ruthenium  Red  (Kuthenium 
Sesquichloride)  (EiSEN,  Zeit.  f.  wiss.  Mik.,  xiv,  2,  1897,  p.  200;  in  my 
hands  totally  useless).  Impregnation  -with  Fats,  Altmann's  Method 
(see post,  "Corrosion"). 


CHAPTER    XIX. 

OTHER  STAINS  AND  COMBINATIONS. 

365,  Kernschwarz  (PLAINER,  Zeit.  f.  wiss.  Mik.,  iv,  3, 
1887,  p.  350;  Journ.  Roy.  Mic.  floe.,  1888,  p.  675).— Kern- 
schwarz is  a  black  liquid  prepared  for  histological  purposes 
by  a  Russian  chemist,  and  imported  by  Griibler  &  Hollborn. 
Its  exact  composition  is  unknown,  but  it  is  certainly  an  ink. 
MAYEE  (Grundzuge,  p.  202)  finds  that  it  contains  a  metallic 
base,  namely  iron,  combined  with  an  organic  acid,  which  is 
highly  probably  some  gallic  acid.  The  liquid  keeps  in- 
definitely, and  affords  a  progressive  or  regressive  stain, 
according  to  circumstances.  I  use  it  as  follows  : 

Sections  (I  have  not  tried  material  in  bulk)  are  fixed  on 
slides  and  treated  with  Kernschwarz  until  they  become  grey 
or  black.  If  the  material  is  fresh,  the  required  depth  of 
stain  may  be  obtained  in  a  few  minutes,  and  in  that  case  it 
may  be  well  to  first  dilute  the  Kernschwarz  with  about  ten 
volumes  of  water  if  it  be  desired  not  to  run  the  risk  of  over- 
staining.  If  the  material  is  not  fresh,  that  is  if  it  has  been 
kept  for  some  months,  it  will  be  necessary  to  stain  for 
twenty-four  hours  in  the  undiluted  liquid. 

There  is  obtained  a  black  or  neutral-tint  stain,  which  is, 
according  to  the  previous  treatment  of  the  material,  either  a 
pure  chromatin  stain,  or  at  the  same  time  a  plasma  stain. 
If  overstaining  should  have  occurred,  or  if  there  has  been 
obtained  a  plasma  stain  that  it  is  desired  to  remove,  the 
stain "  is  easily  differentiated  by  means  of  any  weak  acid, 
either  in  water  or  alcohol.  PLATNER  took  alkalies,  preferably 
carbonate  of  lithia,  for  differentiation  ;  but  that  is  clearly 
faulty  practice. 


OTHER    STAINS    AND  COMBINATIONS.  263 

In  either  case  the  stain  is  an  excellent  one.  The  chroma- 
tin  stain  is  frequently  as  fine  as  any  that  I  know 'of,  except 
that  of  iron-haematoxylin,  resting  and  dividing  nuclei  being 
differentiated  just  as  in  the  best  chromatin  stains. 

If  a  plasma  stain  has  been  obtained,  it  is  generally  a  very 
good  one,  the  cytoplasmic  reticulum  being  well  brought  out, 
and  spindle-relics,  Nebenkerne,  and  other  enclosures  being 
very  well  stained.  It  may  be  well,  if  a  good  plasma  stain 
has  been  obtained,  to  after-stain  for  twenty-four  hours  with 
safraiiin,  followed  by  differentiation  in  either  neutral  or  acid 
alcohol,  and  clove  oil.  This  gives  a  fine  double-stain, 
chromatin  and  nucleoli  being  of  a  dark,  somewhat  brownish 
red,  the  plasma  purple-grey.  The  stain  is  perfectly  per- 
manent in  balsam. 

I  most  highly  recommend  this  stain,  which  is  safe,  easy 
to  carry  out,  and  applicable  to  the  study  of  very  various 
tissues. 

The  stain  is  stated  to  be  a  good  one  for  preparations  that 
it  is  desired  to  photograph. 

366.  Brazilin,  the  colouring  matter  of  Brazilian  redwood  or  Pernam- 
buco  wood,  has  been  recommended  by  EISEX  (Zeit.  f.  wiss.  MiJe.,  xiv,  1897, 
p.  198).     MAYEE  (Grundzuge,  p.  203)  finds  that  it  gives  a  stain  similar  to 
that  of  hjfimatein,  but  much  weaker,  and  is  therefore  at  the  least  super- 
fluous. 

367.  Orchella  (Orseille)  (WEDL,  Arch.  f.  path.  Anat.,  Ixxiv,  p.  143  ; 
Journ.  Hoy.  Mic.  Soc.,  ii,  1879)  :  for  an  account  of  this  substance  vide 
COOLEY'S  Cyclopaedia,  sub  voce  "  Archil ;  "  and  see  FOL,  Lehrb.,  p.  192, 
and  former  editions  of  this  work. 

368.  Orceiu  (!SEAEL,  Virchow's  Archiv,  cv,  1886,  p.  169;  Journ.  Roy. 
Mic.  Soc.,  1887,  p.  514,  and  ISRAEL,  Prakticum  der  path.  Hist.,  2  Aufl., 
Berlin,  1893,  p.  72). — Orcein  is  a  dye  obtained  from  the  tinctorial  lichen, 
Lecanora  parella,  and  is  not  to  be  confused  with  orcin,  another  deriva- 
tive of  the  same  lichen.     It  is  said  to  unite  in  itself  the  staining  properties 
of  the  basic  and   acid  stains,  and  also  the  combination  of   two  contrast 
colours.     Israel  stains  sections  in  a  solution  containing  2  grms.  of  orcein, 
2  grins  of  glacial  acetic  acid,  and  100  c.c.  of  distilled  water,  washes  in 
distilled  water,  and  passes  rapidly  through  absolute  alcohol  to  thick  cedar 
oil,  in  which  the  preparations  remain  definitively  mounted.     Nuclei  blue, 
protoplasm  red. 

For  the  specific  staining  of  elastic  and  connective  tissue  by  means  of  this 
reagent  see  the  paragraphs  on  the  "Connective  Tissues"  in  Part  II. 


264  CHAPTER   XIX. 

369.  Purpurin,   see   KANVIER'S    Traite  technique,  p.  280;    DUVAL'S 
Precis  de  Technique  histologique,  p:  221;  and  GRENACHER'S  formula  in 
Arch.  f.  miJc.  Anat.,  xvi,  1879,  p.  470. 

370.  Indigo. — Indigo  is  employed  in  histology  in  the  form  of  solutions 
of  so-called  indigo-carmine,  or  sulphindigotate  of   soda  or  potash.      The 
simple  aqueous  solution  gives  a  diffuse  stain,  and  is  therefore  not  capable 
of  being  usefully  employed  alone.     It  is,  however,  of  use  when  employed 
to  bring  about  a  double  stain  in  conjunction  with  carmine,  see  below. 

Thiersch's  Oxalic  Acid  Indigo-carmine  (see  Arch.f.  mik.  Anat.,  i, 
1865,  p.  150). 


Carmine  Combinations. 

371.  Seller's     Carmine     followed    by     Indigo  -  Carmine    (Am. 
Quart.  Hie.  Journ.,  i,  1879,  p.  220;    Journ.  Roy.  Hie.  Soc.,  ii,  1879, 
p.  613). — Stain  in  borax-carmine,  wash  out  with  HC1  alcohol,  wash  out  the 
acid,  and  after-stain  in  an  extremely  dilute  alcoholic  solution  of  indigo- 
carmine  (two  drops  of  saturated  aqueous  solution  added  to  an  ounce  of 
alcohol  and  filtered). 

I  find  this  method  gives  good  results  when  applied  to  sections,  but  very 
bad  results  if  it  be  attempted  to  stain  in  the  mass  with  the  indigo.  The 
indigo  overstains  the  superficial  layers  before  it  has  penetrated  to  the 
deeper  layers.  The  instructions  given  refer  to  sublimate  material  or  the 
like ;  I  find  chrom-osmium  material  will  not  take  the  stain  at  all. 

372.  Merkel's    Carmine    and    Indigo- Carmine    in    One    Stain 
(MERKEL,  Unters.  a.  d.  anat.  Anst.  Rostock,  1874;  Month.  Mic.  Journ., 
1877,  pp.  242  and  317). 

Also  NORRIS  and  SHAKESPEARE,  Amer.  Journ.  Med.  Sc.,  January,  1877  ; 
MERKEL,  Mon.  Mic.  Journ.,  1877,  p.  242;  MARSH,  Section  Cutting, 
p.  85 ;  BAYERL,  Arch.  f.  mile.  Anat.,  xxiii,  1885,  pp.  36,  37 ;  MACALLUJI, 
Trans.  Canad.  Instit.,  ii,  1892,  p.  222 ;  Journ.  Roy.  Mic.  Soc.,  v,  1892, 
p.  698. 

MERKEL'S  formula,  as  has  been  pointed  out  by  PAUL  MAYER  (Mitth.  Zool. 
Stat.  Neapel,  xii,  2,  1896,  p.  320)  is  not  only  highly  irrational,  and  incon- 
venient to  employ,  but  gives  an  alkaline  fluid  that  may  be  injurious  to 
tissues.  I  agree  with  him  that  it  should  be  suppressed. 

373.  P.  MAYER'S  Carmalum  (or  Haemalum)  and  Indigo-Car- 
mine in  One  Stain. — In  the  place  quoted  in  the  last  section, 
MAYER  states  that  he  obtains  very  good  results  by  taking  a 
solution  of  O'l  gramme  of  indigo-carmine  in  50  c.c.  of  dis- 
tilled water,  or  5  per  cent,  alum  solution,  and  combining  it 
with  from  four  to  twenty  volumes  of  carmalum  or  haemalum. 


OTHER    STAINS    AND    COMBINATIONS.  265 

374.  Carmine   and  Picro-Indigo-Carmine    (CAJAL,    Rev.    de 
Cienc.   wed.,    1895  ;   CALLEJA,   Rev.   trim.  Microgr.,  ii,   1897, 
p.  101  ;  Zeit.  f.  wiss.  Hik.,  xv,   3,   1899,  p.  323).— For  use 
after  a  carmine  stain,  CAJAL  takes  a  solution  of  0*25  gramme 
of  indigo-carmine  in  100  grammes  saturated  aqueous  solution 
of  picric  acid.      Stain  (section*)  for  five  to  ten  minutes,  wash 
in  weak  acetic  acid,  then  in  water,  then  remove  the  excess 
of  picric  acid  with  absolute  alcohol,  clear  and  mount.      Em- 
ployed in  this  way,  the  indigo  is  said  to  give  a  sharper  plasma 
stain  than  without  the  picric  acid. 

375.  Carmine   and  Anilin   Blue    (or   Bleu   Lumiere,  or  Bleu 
de  Lyon)    (DOVAL,    Precis   de   technique   micro*copique,  1878, 
p.   225) . — Stain    with    carmine    "  in    the   ordinary    way  ;  " 
dehydrate,  and   stain  for  a  few  minutes  (ten  minutes  for  a 
section  of  nerve-centres)  in  an  alcoholic    solution   of  anilin 
blue  (ten  drops  of  saturated  solution  of  anilin  blue  soluble 
in  alcohol  to  ten  grammes  of  absolute  alcohol,  for  sections  of 
nerve-centres).        Clear    with    turpentine,    without    further 
treatment  with  alcohol,  and  mount  in  balsam. 

Recent  authors  recommend,  instead  of  anilin  blue,  bleu  de 
Lyon,  dissolved  in  70  per  cent,  alcohol  acidulated  with  acetic 
acid  (MAURICE  and  SCHULGIN),  or  bleu  lumiere,  which  has 
hardly  any  effect  on  nuclei. 

The  solutions  of  both  these  colours  should  be  extremely 
dilute  for  sublimate  material,  but  strong  for  chrom- osmium 
material.  It  is  possible  to  use  them  for  staining  in  bulk. 

BAUMGARTEN  (Arch.  f.  mik.  Anat.,  xl,  1892,  p.  512J  stains 
sections  (of  material  previously  stained  in  borax-carmine)  for 
twelve  hours  in  a  0'2  per  cent,  solution  of  bleu  de  Lyon  in 
absolute  alcohol,  and  washes  out  for  about  half  that  time 
before  mounting  in  balsam.  He  recommends  the  process  for 
cartilage  and  nerve-centres. 

376.  Carmine  and  Malachite  Green. — HAAS  (Zeit.  /.  wiss.  Zool..  1, 
4,  1890,  p.  527;   Zeit.  f.  wiss.  Mik.,  viii,  2,  1891,  p.  205)  recommends 
borax-carmine  followed  by  weak  alcoholic  solution  of  malachite  green,  with 
a  final  washing  out  with  stronger  alcohol,  see  also  §  302. 

377.  Carmine  and  Picro-nigrosin  (PIANESE)  (see  Journ.  Eoy.  Mic. 
Soc.,  1892,  p.  292). 

378.  Carmine  and  Picric  Acid.     See  §  281. 


266  CHAPTER  XIX. 

Hasmatein  or  Hsematoxylin  Combinations. 

379.  Haematoxylin  and  Picric  Acid. — See  §  281. 

380.  Haematoxylin  and  Eosin  or  Benzopurpurin  (§  295). — 
Objects  may  be  stained  with  haematoxylin  (either  in  the  mass 
or  as  sections)  and  the  sections  stained  for  a  few  minutes  in 
eosin.      I  think  it  is  better  to  take  the  eosin  weak,  though  it 
has  been  recommended  (STOHK,  see  Zeit.f.  wiss.  Milt.,  i,  1884, 
p.    583)   to  take  it  saturated.      Either  aqueous   or    alcoholic 
solutions  of  eosin  may  be  used. 

HICKSON  (Quurt.  Journ.  Mic.  Sci.,  1893,  p.  129)  gives  the 
following  instructions  for  staining  sections  on  the  slide  : — 
One  hour  in  a  strong  solution  of  eosin  in  90  per  cent,  alcohol, 
wash  with  alcohol,  and  stain  for  twenty  minutes  in  a  weak 
solution  of  haematoxylin. 

This  method  is  most  particularly  recommendable  for 
embryological  sections,  as  vitellus  takes  the  eosin  stain  ener- 
getically, and  so  stands  out  boldly  from  the  other  germinal 
layers  in  which  the  blue  of  the  haematoxylin  dominates. 

See  also  LIST  (Zeit.f.  wiss.  Mik.,  ii,  188-%  p.  148)  ;  BUSCH 
(Verh.  Berl.  Pliys.  Ges.,  1887);  GIEKKE  (Zeit.  f.  wis*.  Mik., 
i,  1884,  p.  505). 

It  should  be  noted  that  sections  should  be  very  well  washed 
before  being  passed  from  eosin  into  haematoxylin  or  the  reverse, 
as  eosin  very  easily  precipitates  .haematoxylin. 

381.  RENAUT'S  Haematoxylic  Eosin  (FOL'S  Lehrbuch,  p.  196).  A  very 
complicated  glycerin  mixture,  which  acts  so  slowly  that  it  may  take  weeks 
to  stain,  and,  I  think,  superfluous. 

EVEBAED,  DEMOOE  and  MASSAKT  (Ann.  Inst.  Pasteur,  vii,  1893,  p.  166) 
prepare  a  similar  mixture  as  follows  : — A  solution  is  made  with  1  grm.  of 
eosin,  25  grms.  of  alcohol,  75  of  water,  and  50  of  glycerin.  Then  20  grms. 
of  alum  are  dissolved  by  the  aid  of  heat  in  200  grms.  of  water,  the  solution 
is  filtered,  and  after  twenty-four  hours  there  is  added  to  it  1  grm  of  hsema- 
toxylin  dissolved  in  10  grms.  of  alcohol.  This  solution  is  allowed  to  stand 
for  eight  days,  then  filtered  again,  and  combined  with  an  equal  volume  of 
the  eosin  solution. 

382.  Haematein  and  Congo.— -See  §  294. 


OTHER    STAINS   AND    COMBINATIONS.  267 

383.  Haematein  and  Saurefuchsin. — Get  a  sharp  chromatin 
stain  with  iron-haematoxylin  or  haemalum,  then  stain  (sections) 
in  0*5  per  cent,  aqueous  solution  of  Saurefuchsin,  dehydrate 
and  mount.      The  time  required  for  staining  varies  much  with 
the  material,  but  is  easily  found  by  trial. 

384.  Hsematoxylin  and  Saurefuchsin  and  Orange. —Proceed 
as  above,  using  for  the  second  stain  the  following  mixture  : 
Saurefuchsin,  1  grm.;  orange,  6  grms.;  rectified  spirit,  60  c.c.; 
water,  240  c.c.  (from  SQUIRE'S  Methods  and  Formulas,  p.  42). 
Using  orange  Gr  (not  mentioned  by  SQUIRE),  I  have  had  very 
good  results. 

The  process  described  by  CAVAZZANI  (Riforma  Med., 
Napoli,  1893,  p.  604;  Zeit.  f.  iviss.  Mik.,  xi,  3,  1894,  p.  344) 
is  far  too  complicated  to  be  recommendable. 

385.  Haematoxylin  and  Picro-Saurefucnsin  (VAN  GIBSON,  New 
York  Med.  Journ.,  1889,  p.  57;  quoted  from  MOELLER,  Zeit. 

f.  wiss.  Mik.,  xv,  2,  1898,  p.  172,  which  see  for  further 
details).  Proceed  as  above,  using  for  the  second  stain  the 
picro- Saurefuchsin  mixture,  §  289.  The  second  stain  must  not 
be  too  prolonged  or  the  haematoxylin  stain  may  be  attacked. 
This  stain  is  now  very  much  in  vogue. 

386.  Haematoxylin  and  Safranin. — RABL  (Morph.  Jahrb.,  x,  1884, 
p.  215)  stained  very  lightly  with  vei'y  dilute  DELAFIELD'S  hsematoxylin  for 
twenty-four  hours,  then  for  some  hours  in  (PFITZNEB'S)  safranin,  and 
washed  out  with  pure  alcohol. 


CHAPTER  XX. 

EXAMINATION  AND  PRESERVATION  MEDIA. 

387.  Introductory, — I  comprehend  under  this  heading  all 
the  media  in  which  an  object  may  be  examined.  The  old 
distinction  of  "indifferent"  liquids,  and  those  which  have 
some  action  on  tissues,  appears  to  be  misleading  more  than 
helpful;  inasmuch  as  it  is  now  well  understood  that  no 
medium  is  without  action  on  tissues  except  the  plasma  with 
which  they  are  surrounded  during  the  life  of  the  organism ; 
and  this  plasma  itself  is  only  "  indifferent "  whilst  all  is  in 
situ ;  as  soon  as  a  portion  of  tissue  is  dissected  out  and 
transferred  to  a  slide  in  a  portion  of  plasma  the  conditions 
become  evidently  artificial. 

It  does  not  appear  necessary  to  create  a  separate  group  for 
mounting  media,  as  all  preservative  media  may  be  used  for 
mounting,  though  the  only  media  that  will  afford  an  abso- 
lutely sure  preservation  of  soft  tissues  are  the  resinous  ones. 

For  directions  as  to  making  permanent  mounts  in  fluid 
media  see  the  early  sections  of  Chap.  XXI. 

Watery  Media. 

388.  Water. — To  preserve  it  from  mould,  a  lump  of  thymol  or  camphor 
should  he  kept  in  the  supply.  Water  maybe  employed  without  inconveni- 
ence, and  sometimes  (on  account  of  its  low  index  of  refraction)  with  great 
advantage  for  the  examination  of  all  structures  that  have  been  fixed  with 
osmic  or  chromic  acid,  or  some  salt  of  the  heavy  metals ;  but  it  is  by  no 
means  applicable  to  the  examination  of  fresh  tissues, — that  is,  tissues  that 
have  not  been  so  fixed.  It  is  important  that  the  beginner  should  bear  in 
mind  that  water  is  very  far  from  being  an  "indifferent"  liquid;  many 
tissue  elements  are  greatly  changed  by  it  (nerve-end  structures,  for  in- 
stance), and  some  are  totally  destroyed  by  its  action  if  prolonged  (for 
instance,  red  blood-corpuscles). 


EXAMINATION    AND    PRESERVATION   MEDIA.  269 

389.  Theory   of  Indifferent   Liquids. — In    order    to    render 
water  inoffensive  to  such  tissues  as  these  it  must,  firstly,  have 
dissolved  in  it  some  substance   that  will   give   it    a  density 
equal  to  that  of  the  liquids  of  the  tissue,  so  as  to  prevent  the 
occurrence  of  osmosis,  to  which  process  the  destructive  action 
of  pure  water  is  mainly  due.     Salt  solution  is  a  medium  sug- 
gested  by   this   necessity.       But  salt   solution  by  no  means 
fulfils  all  the  conditions  implied  in  the  notion  of  an  "  indif- 
ferent "  liquid.    In  so  far  as  it  possesses  a  density  approach- 
ing to  that  of  the  liquids  of  the  tissues,  one  cause  of  osmosis 
is  eliminated  ;  but  there  remains  another,  due  to  the  difference 
of  composition  of  the  liquids  within  the  tissues  and  that  with- 
out.   Cell  contents  are  a  mixture  of  colloids  and  crystalloids  ; 
salt  solution  contains  only  a  crystalloid,  whose  high  diffusi- 
bility  causes  it  to  diffuse  over  into  the  colloids  of  the  tissues. 
In   order   to   reduce   the  consequent  osmotic  processes  to  a 
minimum,  it  is  necessary  that  the  examination  medium  con- 
tain, in  addition  to  a  due  proportion  of  salt  or  other  crystal- 
loid,  also    a   due    proportion   of    colloids.       By  adding,    for 
instance,    white    of   egg   to    salt   solution   this   end   may  be 
attained ;  and,  as  a  matter  of  fact,  the  liquids  recommended 
as  indifferent   are  found  invariably  to  contain  both  crystal- 
loids and  colloids.    Thus  (as  stated  by  FREY)  vitreous  humour 
contains  987  parts  of  water  to  about  4'6  of  colloid  matters 
and    7*8   of    crystalloids   (common   salt).      In    1000  parts  of 
liquor  amnii  are  contained  about  3*8  parts  of  colloid  matter 
(albumen) ,  5 '8  of  salt,  and  3*4   of  urea.      In  blood-serum, 
8*5  of  colloids  and  1  of  crystalloid  substance  are  found. 

390.  Salt  Solution  ("  normal  salt  solution/'  "  physiological 
salt   solution"). — 0'75   per  cent,  sodium   chloride  in  water. 
CARNOY  recommends  the  addition  of  a  trace  of  osmic  acid. 

According  to  LOCKE  (Boston  Med.  Surg.  Journ.,  1896,  p.  514)  there 
should  be  added  to  salt  solution  (which  to  be  isotonic  should  contain, 
according  to  HAMBUEGEE,  0'9  to  1  per  cent,  of  salt) — 0*01  per  cent, 
chloride  of  potassium  and  0'02  per  cent,  chloride  of  calcium,  in  order  to 
obtain  an  indifferent  liquid.  MALASSEZ  (C.  R.  Soc.  BioL,  iii,  |896, 
pp.  504  and  511)  takes  for  erythrocytes  about  1  per  cent,  sodium  chloric 

391.  PICTET'S  Liquid  (Mitth.  Zool.  8 tat.  Neapel,  x,  1,  1891, 
p.  89). — 5   to   10  per   cent,   solution   of    chloride    of    man- 


270  CHAPTER    XX. 

ganese.  According  to  my  experience,  this  solution  is  excel- 
lent, and  very  often  advantageously  takes  the  place  of 
t(  normal  salt  solution."  The  proportions  given  are  for 
marine  animals,  and  for  terrestrial  animals  will  generally  be 
found  much  too  high.  For  these  from  1  per  cent,  to  3  per 
cent,  will  be  nearer  the  mark. 

392.  Iodised  Serum. — Iodised  serum  was  first  recommended 
by  MAX  SCHULTZE  (Virchow's  Archiv,  xxx,  1864,  p.  263).  I 
take  the  following  instructions  concerning  it  from  RANVIER 
(Traite,  p.  76). 

The  only  serum  that  gives  really  good  results  is  the  amniotic 
liquid  of  mammals.  A  gravid  uterus  of  a  sheep  or  cow 
having  been  obtained  (in  large  slaughterhouses  such  can  be 
obtained  without  difficulty),  an  incision  is  made  through  the 
wall  of  the  uterus  and  the  foetal  membranes.  A  jet  of  serum 
issues  from  the  incision,  and  is  caught  in  a  flask  prepared  for 
the  purpose.  Flakes  of  iodine  are  then  added,  and  the  flask 
is  frequently  agitated  for  some  days.  Two  points  should  be 
noted.  A  perfectly  fresh  amnios  must  be  taken,  for  the 
merest  incipience  of  putrefaction  will  spoil  the  preparation. 
The  flask  should  have  a  wide  bottom,  so  that  the  serum  may 
form  only  a  shallow  layer  in  it ;  otherwise  the  upper  layers 
will  not  be  sufficiently  exposed  to  the  action  of  the  iodine. 

Another  method  is  as  follows  : — Serum  is  mixed  with  a 
large  proportion  of  tincture  of  iodine ;  the  precipitate  of 
iodine  that  forms  is  removed  by  filtration,  and  there  remains 
a  strong  solution  of  iodine  in  serum.  This  should  be  kept  in 
stock,  and  a  little  of  it  added  every  two  or  three  days  to  the 
serum  that  is  intended  for  use. 

RAN  VIE  K  explains  that  at  the  outset  serum  dissolves  very 
little  iodine  ;  but  if  an  excess  of  iodine  be  kept  constantly 
present  in  the  solution,  it  will  be  found  that  after  two  or 
three  weeks  iodides  are  formed,  and  allow  fresh  quantities  of 
iodine  to  dissolve ;  so  that  after  one  or  two  months  a  very 
strongly  iodised  serum  is  obtained.  It  should  be  dark  brown. 
Such  a  solution  is  the  most  fitting  for  the  purpose  of  iodising 
fresh  serum  in  the  manner  directed  above,  and  for  making 
the  different  strengths  of  iodised  serum  that  are  required  for 
different  purposes.  In  general  for  maceration  purposes  a 
serum  of  a  pale  brown  colour  should  be  employed. 


EXAMINATION    AND    PRESERVATION    MEDIA.  271 

393.  Aqueous  Humour,  Simple  White  of  Egg — Require  no 
preparation  beyond  filtering.      They  may  be  iodised  if  desired. 


394.  Artificial  Iodised  Serum  (FRET,  Das  Mikroskop,  6  Aufl., 
1877,  p.  75). 

Distilled  water     .           .           .           .  270    grins. 

White  of  egg       ....  30 

Sodium  chloride  .           .           .           .  2*5     „ 

Mix,  filter,  and  add  tincture  of  iodine. 

395.  KRONECKER'S    Artificial   Serum  (from   VOGT   et   YUNG, 
Traite  d'Anat.  comp.  prat.,  p.  473). 

Common  salt  .  .  .  .  .          6  grms. 

Caustic  soda  .  .  .  .  .          0*06    grm. 

Distilled  water         ,  1 000  grms. 

BOHM  und  OPPEL  (Taschenbuch,  3  AufL,  p.  19)  take  car- 
bonate of  soda  instead  of  caustic  soda. 

396.  MIGULA'S  Giycerized  Blood-serum  (see  the  paper  in  Zeit.f.  wiss. 
Mik.,  vii,  2,  1890,  p.  172  ;  also  Journ.  Roy.  Mic.  Soc.,  1890,  p.  804). 

397.  Syrup. — An  excellent  medium  for  examining  many 
structures  in  the  fresh  state.  To  preserve  it  from  mould, 
chloral  hydrate  may  conveniently  be  dissolved  in  it  (1  to  5 
per  cent.).  I  have  used  as  much  as  7  per  cent.,  and  found 
no  disadvantage,  or  carbolic  acid  may  be  employed  instead 
of  chloral ;  1  per  cent,  is  sufficient. 

Either  of  these  syrups  may  be  used  as  a  mounting  medium, 
but  they  are  not  to  be  recommended  for  that  purpose,  as  there 
is  always  risk  of  the  sugar  crystallising  out. 

A  good  strength  for  syrup  is  equal  parts  of  loaf  sugar  and 
water.  Dissolve  by  boiling. 

398.  Chloride  of  Calcium  (HABTING,  Das  Mikroskop,  2  Aufl.,  p.  297). 
— The  aqueous  solution,  either  saturated  or  diluted  with  1  to  8  parts  of 
water,  has  a  low  refractive  index  and  does  not  dry  up. 

399.  Acetate  of  Potash  (MAX  SCHULTZE,  Arch.  mik.  Anat.,  vii,  1872, 
p.  180). — A  nearly  saturated  solution  in  water.     The  index  of  refraction  is 
lower  than  that  of  glycerin. 


272  CHAPTER    XX. 

This  medium  has  been  frequently  recommended  as  having  the  property 
of  preventing  the  blackening  of  objects  that  have  been  treated  with  osmium; 
but  it  seems  extremely  doubtful  whether  this  is  really  the  case. 

400.  Chloral  Hydrate. — 5   per   cent,  in  water   (LADOWSKY, 
Arch.f.  mik.  Anat.,  1876,  p.  359). 

Or,  2*5  per  cent,  in  water  (BEADY,  British  Copepods). 
Or,  1  per  cent,  in  water  (MUNSON,  Journ.  Roy.  Mic.  Soc., 
1881,  p.  847). 

401.  Alcohol, — Not    recommeridable    for    mounting,    as    if 
taken  weak  it  is  not  an  efficient  preservative,  and  if  taken 
strong  it  attacks  the  cement  of  mounts. 

The  chief  use  of  alcohol  for  preservation  purposes  is  of 
course  for  preserving  specimens  in  till  wanted  for  further 
preparation  and  study.  See  on  this  point  the  remarks  in 
Chap.  I,  §  3. 

402.  Formaldehyde.— See  §  104. 


Mercurial  Liquids. 

(I  give  these  as  examination  media  only,  not  as  permanent  mounting  media. 
Media  containing  sublimate  always  end  by  making  tissues  granular.) 

403.  GILSON'S  Fluid  (CARNOY'S  Biologie  cellulaire,  p.  94). 
Alcohol  of  60  per  cent.   .  .  .60  c.c. 

Water  .  .  .  .  .      30   „ 

Glycerin         .  .          ..  .  .      30   „ 

Acetic   acid   (15  parts   of  the   glacial 

to  85  of  water) ' .          .  2    „ 

Bichloride     .  .  .  .  0'15  grm. 

A  really  excellent  examination  medium  for  the  study  of 
fine  cellular  detail  with  well-fixed  objects. 


404.    GAGE'S   Albumen  Fluid    (Zeit.   f.   wiss.    Mik.,    1886, 
p.  223). 

White  of  egg          .          .  .          .15  c.c. 

Water 200    „ 

Corrosive  sublimate          .  .  .        0*5  grm. 

Salt      ......        4  grms. 


EXAMINATION    AND  PRESERVATION    MEDIA.  273 

Mix,  agitate,  filter,  and  preserve  in  a  cool  place.  Recom- 
mended for  the  study  of  red  blood-corpuscles  and  ciliated 
cells. 

405.  PACIXI'S  Fluids  (Journ.  de  Mic.,  iv,  1880;  Journ.  Eoy.Mic.  Soc., 
[N.  S.J,  ii,  1882,  p.  702,  and  previous  editions  of  this  work). — Antiquated 
and  superfluous.     They  consist  essentially  of  corrosive  sublimate  of  from 
one  half  to  one  third  per  cent,  strength,  with  the  addition  of  a  little  salt 
or  acetic  acid. 

406.  GOADBY'S  Fluids  (Micro.  Diet.,  art.  "  Preservation,"  or  previous 
editions  of  this  work). — Quite  unsuited  for  histological  purposes. 


Other  Fluids. 

407.  Chloride  and  Acetate  of  Copper  (RIPAET  et  PETIT' s  fluid, 
Brebissonia,  1880,  p.  92;  CARNOY'S  Biol.  cell.,  p.  95). 
Camphor  water  (not  saturated)          .      75       grins. 
Distilled  water      .          .  .      75          „ 

Crystallised  acetic  acid  .  *  .1       grin. 

Acetate  of  copper  .          .          .       0*30    „ 

Chloride  of  copper  .  .          .        0'30    „ 

A  valuable  medium  for  work  with  delicate  fresh  tissues. 
It  may  be  used  in  combination  with  methyl  green,  which  it 
does  not  precipitate.  The  addition  of  a  drop  of  osmic  acid 
or  corrosive  sublimate  does  not  cause  the  least  turbidity,  and 
enhances  its  fixing  action. 

408.  Tannin  (CAENOY,  loc.  cit.). 

Water          .....         100  grms. 

Powdered  tannin     .  .  .  .  0'40  grm. 

As  an  examination  medium  only. 

409.  Methyl  Green. — See  §  277.  The  aqueous  solution  is 
very  useful  as  an  examination  medium  for  fresh  tissues.  It 
should  be  taken  fairly  concentrated,  in  which  state  it  has 
sufficient  fixing  power,  which  is  enhanced  by  the  addition  of 
a  trace  of  osmic  acid. 

410.  WICKEBSHEIMER'S  Fluid  (Zool.  Anz.,  1879,  p.  670;  cf.  Jowrn. 
Roy.  Mic.  Soc.,  1882,  p.  427;  id.,  1880,  p.  355;  and  Entomol.  Nachr., 
1880,  p.  129).— Worthless  for  histological  purposes. 

18 


274  CHAPTtilt  XX. 

411.  MEYER'S  Salicylic  Vinegar  Preservative  Solutions  (Arch, 
mik.  Anat.,  xiii,  1876,,  p.  868). — Solution  of  salicylic  acid  in 
pyroligneous  acid  and  glycerin.      See  previous  editions. 

412.  NOLI/S  Salicylic  Vinegar  and  Gum  Medium  (Zool.  An?.., 
1883,  p.  472). — A  mixture  of  MEYEK'S  fluid  and  medium  of 
FAERANTS.      See  previous  editions. 

413.  DEANE'S  Medium  (see  Micro.  Diet.,  art.  "Preservation  "). 

414.  HOYEK'S  Gum  with  Chloral  Hydrate  or  Acetate  of  Potash 
(Biol.  Centratt.,  ii,  1882,  pp.  23-4;  Journ.  Roy.  Mic.  Soc.  [N.  S.],  iii, 
1883,  pp.  144-5). — A  high  60  c.c.  glass  with  a  wide  neck  is  filled  two  thirds 
full  with  gum  arabic  (in  pieces),  and  then  either  a  solution  of  chloral  (of 
several  per  cent.)  containing  5 — 10  per  cent,  of  glycerin  is  added,  or  officinal 
solution  of  acetate  of  potash  or  ammonia.     The  gum  with  frequent  shaking 
dissolves  in  a  few  days,  and  forms  a  syrupy  fluid,  which  is  slowly  filtered 
for  twenty-four  hours.     The  clear  filtered  fluid  will  keep  for  a  longtime, 
but  if  spores  of  fungi  begin  to  develop  a  little  chloral  can  be  added  and  the 
fluid  refiltered.     The  solution  with  chloral  is  for  carmine  or  hsematoxylin 
objects — that  with  acetate  for  anilin  objects. 

415.  Medium  of  FARRANTS  (BEALE,  How  to  Work,  etc.,  p.  58). 
Picked  gum  arabic  .  '.          .  .4  ounces. 

Water    .  .         '.          .'         .  .      4 

Glycerin  .    .          ..          .  .2        „ 

To  be  kept  in  a  stoppered  bottle  with  a  lump  of  camphor. 
Slightly  different  receipts  for  this  are  given  by  the  Micro- 
graphic  Dictionary,  and  A.  F.  STANLEY  KENT,  in  Journ.  Eoy. 
Mic.  Soc.,  1890,  p.  820. 

416.  Gum  and  Glycerin  Medium  (LANGEKHANS,  Zool.  Anzeig.,  ii 
1879,  p.  575). 

Gummi  arab.  .  .  .  .  .     5'0 

Aquse  .  .  .  .  .  .     5'0 

To  which  after  twelve  hours  are  added — 

Glycerin!        .  .  .  .  .5*0 

Sol.  aquosa  acid,  carbol.  (5*100)  .  .  .  10  0 

417.  FAEIS'S  Glycero-gum  (The  Microscope,  x,  1890,  p.  59;  Journ. 
Eoy.  Mic.  Soc.,  1890,  p.  414).— Gum  arabic  2  ounces,  glycerin  1'5  ounces, 
water  1'5  ounces,  thymol  1  grm.     Mix,  dissolve  with  heat,  and  filter. 

418.  Gum  and  Glycerin  Jelly  (SHIMEE,  The  Microscope,  ix,  1889, 


EXAMINATION   AND    PEESERVATION    MEDIA.  275 

p.  138;  Journ.  Roy.  Mic.  Soc.,  1890,  p.  411).— Equal  parts  of  glycerin 
jelly  (FoL's  second  formula,  post,  §  432),  Farrants'  solution,  and  glycerin. 

419.  COLE'S  Gum  and  Syrup  Medium.     See  §  178. 

420.  APATHY'S  Gum  and  Syrup  Medium  (see  §  328). — This 
medium  is  recommended  by  APATHY  in  a  general  way,  and 
not  merely  for  the  special  purpose  for  which  it  is  quoted  in 
§  328.      It  sets  very  hard,  and,  combined  with  a  paper  cell 
(see  §  453),  may  be  used  for  ringing  glycerin  mounts.- 

421.  FABRE-DOMERGUE'S  Glucose   Medium  (La  Nature,  No. 
823,  9  Mars,  1889,  supp.). 

Glucose  syrup  diluted  to  twenty- 
five  degrees  of  the  areometer 
(sp.  gr.  1-1968)  .  .  1000  parts. 

Methyl  alcohol        .  .  .        200      „ 

Glycerin         .  .  .  100      „ 

Camphor  to  saturation. 

The  glucose  is  to  be  dissolved  in  warm  water,  and  the 
other  ingredients  added.  The  mixture,  which- is  always  acid, 
must  be  neutralised  by  the  addition  of  a  little  potash  or 
soda. 

This  medium  is  said  to  preserve  without  change  almost  all 
animal  pigments. 

421a.  BRUN'S  Glucose  Medium  (from  FABRE-DOME  ROUE'S  Pre- 
miers Principes  du  Microscope  et  de  la  Technique  micro- 
scopique,  Paris,  1889,  p.  123). 

Distilled  water    ....      140  parts. 
Camphorated  spirit       .  .  .        10     „ 

Glucose      .          .          .          .  40     „ 

Glycerin     .  .  .  .  .        10     „ 

Mix  the  water,  glucose,  and  glycerin,  then  add  the  spirit, 
and  filter  to  remove  the  excess  of  camphor  which  is  pre- 
cipitated on  mixing.  Dr.  HENNEGUY  informs  me  that  this 
liquid  is  often  preferable  to  glycerin,  because  it  preserves  the 
colour  of  preparations  stained  with  anilin  dyes,  methyl  green 
included. 

422.  Levulose  is  recommended  as  a  mounting  medium  by  BEHBEN>. 
KOSSEL,  u.  SCHIEFFERDECKEB  (Das  Mikroskop  u.  d.  Meth.  d.  mik.  Unters., 


276  CHAPTER  XX. 

Braunschweig,  1889).  It  is  uncrystallisable,  and  preserves  well  carmine 
and  coal-tar  stains  (hsematoxylin  stains  fade  somewhat  in  it).  The  index 
of  refraction  is  somewhat  higher  than  that  of  glycerin.  Objects  may  be 
brought  into  it  out  of  water. 


Glycerin  Media. 

423.  Glycerin. — Glycerin  diluted  with  water  is  frequently 
employed  as  an  examination  and  mounting  medium.  Dilution 
with  water  is  sometimes  advisable  on  account  of  the  increased 
visibility  that  it  gives  to  many  structures  by  lowering  the 
index  of  refraction  of  the  glycerin.  But  from  the  point  of 
view  of  efficacious  preservation  it  is  always  advisable  to  use 
undiluted  glycerin,  the  strongest  that  can  be  procured. 

Long  soaking  of  tissues  in  glycerin  of  gradually  increased 
strength  is  a  necessary  preliminary  to  mounting  in  all  cases 
in  which  it  is  desired  to  obtain  the  best  possible  preparations, 
and  to  ensure  that  they  shall  keep  well.  If  this  soaking  is 
done  on  the  slide  (the  cover  being  removed  and  the  object 
treated  with  fresh  glycerin  every  one  or  two  days),  it  is  well 
to  take  the  precaution  recommended  by  BEALE,  of  luting  the 
edges  of  the  cover  so  as  to  make  the  preparation  air-tight,  as 
glycerin  is  so  highly  hygroscopic  that  a  drop  of  it  exposed 
to  the  air  rapidly  diminishes  in  strength  to  a  very  con- 
siderable degree.  In  order  to  facilitate  the  removal  of  the 
cover  in  this  process,  the  slide  may  be  gently  warmed  by 
passing  it  two  or  three  times  through  the  flame  of  a  spirit 
lamp.  No  preparation  can  be  considered  to  be  made 
secundum  artem  until  every  part  of  the  object  has  been 
thoroughly  impregnated  with  strong  pure  glycerin. 

For  closing  glycerin  mounts,  the  edges  of  the  cover 
should  first  (after  having  been  cleansed  as  far  as  possible 
from  superfluous  glycerin)  be  painted  with  a  layer  of  glycerin 
jelly ;  as  soon  as  this  is  set  a  coat  of  any  of  the  usual 
cements  may  be  applied.  See  next  Chapter. 

Glycerin  dissolves  carbonate  of  lime,  and  is  therefore  to 
be  rejected  in  the  preparation  of  calcareous  structures  that 
it  is  wished  to  preserve. 

424.  Extra-refractive  Glycerin.— The  already  high  index  of  refrac- 
tion of  glycerin  (Price's  glycerin,  n  =  T46)  may  be  raised  to  about  that  of 


EXAMINATION    AND    PRESERVATION    MEDIA.  277 

crown  glass  by  dissolving  suitable  substances  in  the  glycerin.  Thus  the 
refractive  index  of  a  solution  of  chloride  of  cadmium  (CdCU)*  in  glycerin 
may  be  1'504;  that  of  a  saturated  solution  of  sulphocarbolate  of  zincf  in 
glycerin  may  be  I'oOl ;  that  of  a  saturated  solution  of  SCHEEING'S  J  chloral 
hydrate  (in  crusts)  in  glycerin  is  1-510;  'that  of  iodate  of  zinc  in  glycerin 
may  be  brought  up  to  1'56.§  The  clearing  action  of  glycerin  may  thus  be 
greatly  increased,  and  the  full  aperture  of  homogeneous  objectives  brought 
to  bear  on  objects  mounted  in  one  of  the  above-named  solutions.  For 
further  details  see  previous  editions. 

425.  BAKFF'S  Boroglyceride  (see  Journ.  Roy.  Mic.  Soc,,  1882,  p.  124). 
—This  preparation  may  be  obtained  (price  Is.  per  bottle)  from  The  Kreo- 
chyle  Company,  Viaduct  House,  Farringdon  Street,  E.G.,  or  all  wholesale 
chemists. 

426.  Glycerin  and  Alcohol  Mixtures. — These  most  useful 
fluids  afford  one  of  the  best  means  of  bringing  delicate 
objects  gradually  from  weak  into  strong  glycerin.  The 
object  is  mounted  in  a  drop  of  the  liquid,  and  left  for  a  few 
hours  or  days,  the  mount  not  being  closed.  By  the  evapora- 
tion of  the  alcohol  the  liquid  gradually  increases  in  density, 
and  after  some  time  the  mount  may  be  closed,  or  the  object 
brought  into  pure  glycerin  or  glycerin  jelly. 

1.  CALBERLA'S    LIQUID    (Zeit.  v;iss.    ZooL,   xxx,    1878,    p. 
442). 

Glycerin  .  .  .  .  .1  part. 

Alcohol  .  .  .  .  .2  parts. 

Water 3      „ 

As  already  pointed  out  (§  2),  this  liquid  is  in  many  cases 
to  be  preferred  to  alcohol  for  keeping  fixed  objects  in  until 
required  for  dissection  or  other  further  preparation. 

2.  I  strongly  recommend  the  following  for  very  delicate 
objects : 

Glycerin          ...                      .1  part. 
Alcohol            .          .          .          .          .      1      „ 
Water 2  parts. 

3.  HANTSCB'S  LIQUID. 

Glycerin  .  .  .  .  .1  part. 

Alcohol 3  parts. 

Water -      2      „ 

*  Journ.  Roy.  Mic.  Soc.,  ii,  1879,  p.  346. 
f  Ibid.,  iii,  1880,  p.  1051. 
+  Ibid.  (N.  S.),  i,  1881,  p.  9i:i. 
§  Ibid.,  p.  366. 


278  CHAPTER    XX. 

4.  JAGER'S  LIQUID  (YoGT  and  YUNG'S  Traite  d'Anat.  comp. 
prat.,  p.  16). 

Glycerin  .  .  .  .  .1  part. 

Alcohol  .  .  .  .  1      „ 

Sea  water       .          .          .          .          .10  parts. 

Glycerin  Jellies. 

427.  DEANE'S  Glycerin  Jelly  (from  FREY'S    Le  Microscope, 
p.  231). — 120   grammes   glycerine,   60    grammes   water,    30 
grammes   gelatin.      Dissolve  the   gelatin   in   the  water,  and 
add  the  glycerin.      This,  and  the  following  glycerin  jellies, 
must  of  course  be  used  warm. 

428.  LAWRENCE'S  Glycerin  Jelly  (DAVIES,  Preparation  and 
Mounting  of  Microscopic  Objects,  p.  84). — "  He  takes  a  quan- 
tity of  Nelson's  gelatin,  soaks  it  for  two  or  three  hours  in 
cold  water,  pours  off  the  superfluous  water,   and  heats  the 
soaked    gelatin   until   melted.      To    each  fluid  ounce  of  the 
gelatin,  whilst  it  is  fluid  but  cool,  he  adds  a  fluid  drachm  of 
the  white  of  an  egg.      He  then   boils  this  until  the  albumen 
coagulates  and  the  gelatin  is  quite  clear,  when  it  is  to  be 
filtered  through  fine  flannel,  and  to  each  ounce  of  the  clari- 
fied solution  add  6  drachms  of  a  mixture  composed  of  1  part 
of  glycerin  to  2  parts  of  camphor  water." 

429.  BEALE'S  Glycerin  Jelly  (How  to  Work,  etc.,  p.  57). — 
Gelatin  or  isinglass,  soaked,  melted,  and  clarified  if  desired, 
as  in  the  last  formula.      To  the   clear  solution  add  an  equal 
bulk  of  strong  glycerin. 

430.  BRANDT'S  Glycerin  Jelly  (Zeit.  /.  wiss.  Mik.,  ii,  1880, 
p.  69;   Journ.   Roy.  Mic.  Soc.,   iii,    1880,   p.    502).— Melted 
gelatin  1  part,  glycerin  1J  parts. 

The  gelatin  to  be  soaked  in  water  and  melted  in  the  usual 
way.  After  incorporating  the  glycerin,  the  mixture  is  to  be 
filtered.  This  is  a  point  of  vital  importance,  as  the  gelatin 
of  commerce  is  always  mixed  with  particles  of  dust  and 
minute  threads.  Swedish  filtering  paper  does  not  allow  the 
fluid  to  pass  through  sufficiently,  and  flannel  produces  more 
threads  than  before.  BRANDT  filters  through  spun  glass 
pressed  into  the  lower  part  of  a  funnel.  He  describes  a 


EXAMINATION    AND    PRESERVATION    MEDIA.  279 

simple  arrangement  for  keeping  the  funnel  warm  during  the 
filtering  (see  previous  editions] . 

Some  drops  of  carbolic  acid  should  be  added  to  the  fluid 
product  of  the  filtering. 

431.  KAISER'S  Glycerin  Jelly  (Bot.  Cent.,  i,  1880,  p.   25  ; 
Journ.  Roy.  Hie.  Soc.,  iii,  1880,  p.  504). — One  part  by  weight 
finest  French  gelatin  is  left  for  two  hours  in  6  parts  by  weight 
distilled  water,  7  parts  of  glycerin  are  added,  and  for  every 
100  grammes  of  the  mixture  1  gramme  of  concentrated  car- 
bolic acid.      Warm  for  ten  to  fifteen  minutes,  stirring  all  the 
while,  until  the  whole  of  the  flakes  produced  by  the  carbolic 
acid  have  disappeared.      Filter  whilst  warm  through  the  finest 
spun  glass  laid  wet  in  the  funnel. 

I  prepared  some  of  this  jelly  many  years  ago,  and  find  it 
is  still  perfectly  clear. 

432.  FOL'S  Glycerin  Jellies  (Lehrb.,  p.  138). 

1.  Melt  together  one  volume  of  Beale's  jelly  (§  429)  and 
one  half  to  one  volume  of  water,  and  add  2  to  5  per  cent,  of 
salicylic  acid  solution,  or  carbolic  acid  or  camphor. 

2.  Gelatin 30  parts. 

Water  .  .  .  .          .  70     „ 

Glycerin 100      „ 

Alcoholic  solution  of  camphor  .  5      „ 

Prepare  as  before,  adding  the  camphor  last. 

3.  Gelatin 20  parts. 

Water  .  .        •   .          .          .    150     „ 

Glycerin 100     „ 

Alcoholic  solution  of  camphor  .      15     „ 

433.  SQUIRE'S     Glycerin     Jelly     (SQUIRE'S    Methods     and 
Formula,  etc.,  p.  84).— Soak  100  grms.  of  French  gelatin  in 
chloroform  water,  drain  when  soft,  and  dissolve  with  heat  in 
750  grms.  of  glycerin.      Add  400  grms.  of  chloroform  water 
with  which  has  been  incorporated  about  50  grms.  of  fresh 
egg-albumen ;  mix  thoroughly,  and  heat  to  boiling-point  for 
about  five  minutes.      Make  up  the  total  weight  to  1550  grms. 
with  chloroform  water.      Filter  in  a  warm  chamber. 

434.  GILSON'S  Chloral  Hydrate  Jelly  (communicated  by  Prof. 
GILSON). — 1   vol.    of   gelatin,   melted   secundum    artem,    and 


280  CHAPTER   XX. 

1  vol.  of  Price's  glycerin.  Mix,  and  add  1  vol.  of  chloral 
hydrate  (i.e.  add  crystals  of  chloral  until  the  volume  of  the 
mixture  has  increased  by  one  half)  ;  warm  till  dissolved. 
This  gives  a  very  highly  refractive  aqueous  mounting  medium, 
which  is  found  useful  for  opaque  tissues  that  it  is  desired  not 
to  dehydrate. 

A  similar  medium  is  published  by  GEOFFROY,  Journ.  de 
Botan.,  1893,  p.  55  (see  Zeit.  f.  wiss.  Mik.,  ix,  4,  1893, 
p.  476).  He  dissolves,  by  the  aid  of  as  little  heat  as  pos- 
sible, 3  to  4  grms.  of  gelatine  in  100  c.c.  of  10  per  cent, 
aqueous  solution  of  chloral  hydrate. 


High  Refractive  Liquids. 

435.  STEPHENSON'S  Biniodide  of  Mercury  and  Iodide  of  Potas- 
sium (Journ.  Roy.  Mic.  Soc.  [N.  S.],  ii,  1882,  p.  167).— A  solu- 
tion of  the  two  salts  in  water.  "  This  is  very  easily  prepared 
by  adding  the  two  salts  to  the  water  until  each  shall  be  in 
excess ;  when  this  point  of  saturation  has  been  reached  the 
liquid  will  be  found  to  have  a  refractive  index  of  1'68,  by 
far  the  highest  of  any  aqueous  solution  known  to  me." 
Any  lower  index  can  be  obtained  by  suitable  dilution  with 
water. 

This  fluid  is  very  dense,  its  specific  gravity  being  3*02. 
It  is  highly  antiseptic. 

<l  For  marine  aoimals  a  weak  solution  is  probably  well 
adapted,  as  about  a  1  per  cent,  solution  (5  minims  to  the 
ounce)  will  give  the  specific  gravity  of  sea  water,  with  no 
appreciable  difference  in  the  refractive  index/' 

Covers  should  be  sealed  with  white  wax,  and  the  mounts 
finished  with  two  or  three  coatings  of  gold-size  and  one  of 
shellac. 

I  have  experimented  both  with  strong  and  with  weak 
solutions.  They  are  not  adapted,  I  find,  for  the  purposes  of 
a  permanent  mounting  medium.  Tissues  are  wrell  preserved, 
but  the  preparations  are  ruined  by  a  precipitate  which  forms 
in  the  fluid.  But  as  a  temporary  examination  medium  I 
have  occasionally  found  this  solution  valuable.  Its  optical 
properties  are  wonderful ;  it  allows  of  the  examination  of 
watery  tissues,  without  any  dehydration^  in  a  medium  of 


EXAMINATION    AND    PRESERVATION   MEDIA.  281 

refractive   index    surpassing    that    of    any   known    resinous 
medium. 

See  further  details  in  previous  editions. 

436.  Monobromide  of  Naphthalin. — See  Joum.  Roy.  Hie.  Soc., 
-1880,  p.  1043  (ABBE  and  VAN  HEURCK),  and  Zool  Anz.,  1882,  p.  555  (MAX 
FLESCH). 

437.  THOMPSON'S  High  Refractive  Medium. — See  Journ.  Roy.  Mic. 
Soc,,  1892,  p.  902. 


Resinous  Media. 

438.  Resins  and  Balsams. — Resins  and  balsams  consist  of  a 
vitreous  or  amorphous  substance  held  in  solution  by  an  essen- 
tial oil.  By  distillation  or  drying  in  the  air  they  lose  the 
essential  oil  and  pass  into  the  solid  state.  It  is  these  solidi- 
fied resins  that  should,  in  my  opinion  (and  that,  I  believe,  of 
the  best  microscopists),  be  employed  for  microscopical  pur- 
poses ;  for  the  raw  resins  always  contain  a  certain  proportion 
of  water,  which  makes  it  difficult  to  obtain  a  clear  solution 
with  the  usual  menstrua,  is  injurious  to  the  optical  properties 
of  the  medium  and  to  its  preservative  qualities,  and,  further, 
especially  hurtful  to  the  preservation  of  stains.  I  therefore 
do  not  share  the  contrary  opinion  expressed  by  FOL  (Lehrb., 
pp.  138-9),  but  recommend  that  all  solutions  be  made  by 
heating  gently  the  balsam  or  resin  in  a  stove  until  it  becomes 
brittle  when  cold,  and  then  dissolving  in  an  appropriate 
menstruum.  Solid  resins  are  now  easily  found  in  com- 
merce. 

Solutions  made  with  volatile  menstrua,  such  as  xylol  and 
chloroform,  set  rapidly,  but  become  rapidly  brittle.  Solutions 
made  with  non-volatile  media,  such  as  turpentine,  set  much 
less  rapidly,  and  pass  much  less  rapidly  into  the  brittle  state. 
The  former  should,  therefore,  be  employed  whenever  it  is 
desired  to  have  a  mount  that  sets  hard  rapidly  ;  but  the 
latter  should  be  employed  whenever  it  is  above  all  desired  to 
have  a  mount  that  will  prove  as  durable  as  possible. 

According  to  my  experience,  there  is  no  auch  thing  as  a 
faultless  resinous  mounting  medium  for  histoloyical  purposes. 
Solutions  of  gum  damar  in  xylol  are  very  beautiful  from  the 


282  CHAPTER  XX. 

physical  point  of  view,  and  frequently  afford  a  better  defini- 
tion of  delicate  detail  than  Canada  balsam  does.  But  I 
very  strongly  suspect  that  no  da-mar  solution  is  perfectly 
stable.  A  review  of  some  old  damar  mounts  has  shown  that 
the  majority  of  them  have  developed  granules  that  have 
deteriorated  the  preparations  to  a  greater  or  less  extent. 
(These  granules  are  in  the  worst  cases  large  enough  to  at 
once  attract  attention  even  with  low  powers ;  at  other  times 
they  are  so  small  that  they  can  only  be  seen  with  the  highest 
powers,  and  in  this  case  may  be  mistaken  for  normal  elements 
of  cells.)  Xylol-balsam  and  benzol-balsam  mounts  are  in 
the  same  case,  but  to  a  less  degree.  Chloroform  balsam 
keeps  much  better,  so  far  as  granules  are  concerned.  But 
it  becomes  very  brown  with  a#e,  and  has  the  defect  that  it 
is  injurious  to  stains  made  with  coal-tar  colours.  Seiler's 
alcohol-balsam  keeps  remarkably  well,  but  it  also  will  not 
preserve  the  coal-tar  stains.  For  these  and  other  stains  I 
now  often  use  turpentine  colophonium.  It  gives  very  good 
definition  of  delicate  detail,  and  keeps  perfectly.  (Dr.  PAUL 
MAYEK,  however,  writes  me  that  turpentine  solutions  are  not 
at  all  good  for  alum-haematein  stains.)  Turpentine  colo- 
phonium has  a  rather  low  index  of  refraction  for  objects 
that  require  much  clearing.  For  these  I  very  frequently 
use  oil  of  cedar  wood  in  preference  to  any  resinous  medium. 
It  keeps  perfectly.  With  time  it  thickens  sufficiently  to 
hold  the  cover  in  place  ;  or,  if  desired,  preparations  may  be 
luted  with  BELL'S  cement.  After  using  an  oil  immersion 
objective  011  a  fresh  mount,  it  is  always  easy  to  change  the 
cover  by  floating  it  up  with  a  drop  of  the  oil  placed  at  the 
side. 

Another  reason  for  preferring  turpentine  colophonium, 
where  possible,  is  that  it  does  not  shrink  in  drying  nearly  so 
much  as  the  media  made  with  volatile  solvents. 

Still  another  motive  is  that  turpentine  media  preserve  the 
index  of  visibility  of  the  preparations  much  longer  than  do- 
media  made  with  volatile  menstrua.  Preparations  made  with 
these  last  become  so  transparent  in  course  of  time  that  much 
fine  detail  is  often  lost.  (Such  mounts  may,  however,  be 
revivified  without  removing  the  cover  by  putting  them  for  a 
day  or  two  into  a  tube  of  benzol ;  the  benzol  penetrates  the 
balsam,  and  brings  it  down  to  a  lower  refractive  index.) 


EXAMINATION    AND    PRESERVATION    MEDIA.  283 

The  risibility  of  minute  structures  is  proportional  to  the 
<Hn''  rence  between  the  refractive  indices  of  the  object  and  of 
the  medium  in  which  it  is  mounted.  The  majority  of  the 
elements  of  soft  tissues  are  (after  fixation)  of  an  index  of 
refraction  somewhat  superior  to  that  of  Canada  balsam.  It 
follows  that  by  lowering  the  index  of  the  balsam,  increased 
visibility  is  obtained,  and  the  desideratum  in  any  case  is  to 
find  a  medium  just  low  enough  to  give  good  visibility,  and 
yet  not  so  low  as  to  seriously  cut  down  the  N.A.  of  the  objec- 
tives employed. 

439.  Choice   of  a   Mounting   Medium. — For    the    foregoing 
reasons    I    recommend    turpentine   colophonium   for    general 
work,    with   the   restrictions   mentioned,    §  443 ;    whilst    for 
cases  in  which  a  more  highly  refractive  medium  is  desired, 
I  would  recommend  oil  of  cedar  or  xylol-balsam  for  coal-tar 
stains.    Xyiol-balsam  is  certainly  a  very  fine  medium.    I  have 
merely  wished  to  point  out  that  it  is  not  perfectly  safe,  on  the 
score  of  the  possible  formation  of  granules.      (P.  MAYER,  in 
Utt.,  is  of  the  same  opinion.)      I  also  recommend  for  carmine 
or  iron-haematoxylin  stains   SEILER'S  alcohol-balsam   (MAYER 
prefers  VOSSELER'S  turpentine,  §  444).      For  haemalum  stains, 
xylol-balsam. 

440.  Canada  Balsam. — Prepare   with   the   solid  balsam    as 
above    described,    §    438.      The    usual   menstrua    are   xylol, 
benzol,  chloroform,  and  turpentine.    Dissolve  the  solid  balsam 
in  one  of  these  to  the  required  consistence.      The  turpentine 
solution  is  to  be  preferred  only  in  cases  where  it  is  desired 
to  have  a  medium  that  sets  very  slowly.      (The  objection  to 
turpentine    as   a   solvent  is   that  it  does  not  always  give  a 
homogeneous   solution  with  Canada   balsam  as  it  does  with 
colophonium.)     For  most  other  purposes  the  xylol  solution  is 
the  best.      If  time  be  an  object,  a  benzol  solution  should  be 
preferred,  as  it  sets  much  quicker  than  the  xylol  solution. 

SAHLI  (Zeit.  f.  u-iss.  Mik.,  ii,  1885,  p.  5)  dissolves  in  cedar 
oil. 

Samples  of  balsam  that  are  acid  are  frequently  met  with,  and  are 
injurious  to  some  stains.  Griibler  &  Hollborn  now  prepare  a  neutral 
balsam,  in  which  MAYER  has  found  that  very  delicate  preparations,  that 
lost  colour  immediately  in  any  other  sort  of  balsam,  have  kept  perfectly  for 


284  CHAPTER  XX. 

many  months.    For  a  process  of  neutralising  balsam  with  carbonate  of  soda 
or  potash  see  COLUCCI  (Giorn.  Ass.  Med.  Natural.  Napoli,  vii,  1897,  p.  172). 

441.  SEILER'S  Alcohol  Balsam  (Proc.  Amer.  Soc.  Mic.,  1881, 
pp.  60-2;  Journ.  Eoy.Mic.8oc.  [N.  S.],ii,  1882,  pp.  126-7).— 
"  Take  a  clear  sample  of  Canada  balsam  and  evaporate  it  in 
a  water-  or  sand-bath  to  dryness ;  i.  e.  until  it  becomes  brittle 
and  resinous  when  cold.      Dissolve  this  while  Avarm  in  warm 
absolute  alcohol,  and  filter  through  absorbent  cotton." 

The  advantage  of  this  medium  is  stated  to  be  that  objects 
may  be  mounted  in  it  direct  from  absolute  alcohol,  without 
previous  treatment  with  an  essential  oil  or  other  clearing 
agent ;  SEILBR  considers  that  by  this  means  "  shrivelling'  is 
avoided,  as  well  as  the  solution  of  fat  in  the  cells." 

The  process  of  mounting  direct  from  alcohol  is  not  very 
easy  to  carry  out,  and  I  cannot  recommend  it  for  general 
work.  But  used  in  the  ordinary  way,  after  clearing  by  an 
essence,  or  byxylol  or  the  like,  SEILER'S  solution  is  for  many 
purposes  admirable. 

As  stated  above,  I  find  that  it  is  one  of  the  most  stable 
solutions  known  to  me.  (My  stock,  made  up  fifteen  years 
ago,  is  still  perfectly  limpid,  and  has  not  darkened  in  colour 
to  an  injurious  extent.)  It  works  pleasantly  enough  (if  care 
be  taken  not  to  breathe  on  it  during  the  process  of  mounting, 
as  this  may  easily  cause  cloudiness).  The  definition  is  very 
fine,  and  the  preservation  of  the  preparations  almost  in- 
variably perfect ;  my  oldest  preparations  only  show  a  few 
granules  of  little  importance.  Of  course  it  has  the  limitation 
that  it  cannot  be  used  with  the  soluble  coal-tar  colours. 

442.  Damar  (Gum   Damar,  or  Dammar,  or  d'Ammar). — The 
menstrua  are  the  same  as  for  balsam,  and  the  solution  should 
be  prepared  in   the  same  way.      The  most  beautiful   of  all 
these   mounting  media  is  the    solution    of   damar  in   xylol. 
Heat  is  not  necessary  to  make  the  solution. 

Minute  directions  (which  I  think  unnecessary)  for  preparing 
a  working  solution  are  given  by  MARTI  NOTTI  in  Zeit.  f.  iciss. 
Mik.,  iv,  2,  1887,  p.  156,  and  in  Malpighia,  ii,  1888,  p.  270  ; 
rf.  also  Journ.  Roy.  Mic.  Soc.,  1889,  p.  163. 

FLKMMING,  PPITZNER,  and  a  writer  signing  C.  J.  M.,  all 
employ  a  mixture  of  benzol  and  turpentine  (see  Arch.  mik. 


EXAMINATION  AND    PRESERVATION    MEDIA. 

Anat.,  xix,  1881,  p.  322;  Sci.  Gossip,  1882,  p.  257;  Journ. 
Roy.  Hie.  Soc.  [N.  S.],  iii,  1883,  p.  145;  Morphol.  Jahrb.,  vi, 
1880,  p.  469 ;  Journ.  Roy.  Mic.  Soc.  [N.  S.],  ii,  1882,  p.  583). 

See  further  details  concerning  these  and  other  solutions  in 
former  editions. 

I  quite  acknowledge  the  special  beauty  of  definition 
obtained  by  means  of  damar  solutions,  but  I  am  convinced 
that  not  one  of  these  solutions  can  be  depended  on  for  really 
permanent  preservations.  Sooner  or  later,  sometimes  after  a 
few  weeks  or  days,  or  it  may  be  only  after  months  or  years 
the  granules  mentioned  in  §  438  will  make  their  appearance. 

443.  Colophonium.— A  solution  of  colophonium  in  turpen- 
tine was  first  recommended  by  KLEINENBEEG.  I  find  it  to  be 
most  highly  recommendable. 

This  medium  sets  very  slowly,  so  that  ample  time  is  afforded 
for  arranging  objects  in  it.  Both  KLEINENBERG  and  MAYER 
warn  against  the  employment  of  absolute  alcohol  as  a  solvent ; 
the  preparations  are  beautiful  at  first,  but  soon  become 
spoiled  by  the  precipitation  of  crystals  or  of  an  amorphous 
substance. 

The  turpentine  solution  keeps  perfectly  limpidj  gives  very 
good  definition,  and  is  altogether  so  excellent  a  medium  that 
I  am  surprised  that  it  is  not  more  used.  It  should  be  recom- 
mended to  beginners.  And,  as  stated  in  §  438,  I  consider 
that  for  many  purposes  it  is  perhaps  the  best  and  most  reliable 
medium  known.  To  make  the  solution,  I  add  small  lumps  of 
colophonium  to  a  quantity  of  rectified  oil  of  turpentine  kept 
in  a  stove,  and  when  a  sufficiently  thick  solution  has  been 
obtained,  filter  twice,  the  filtering  being  done  in  the  stove. 
About  a  fortnight  is  required  for  the  whole  process.  The 
solution  should  not  be  too  thick,  as  it  thickens  somewhat 
with  age.  The  palest  sorts  of  colophonium  should  of  course 
be  selected. 

Of  course  the  slowness  of  drying  of  this  medium  is  a  great 
objection  to  its  use  in  cases  in  which  it  is  required  to  study 
the  preparations  with  oil-immersion  lenses  as  soon  as  possible 
after  mounting.  In  the  winter  a  slide  will  take  about  a 
month  before  it  will  be  hard  enough  to  be  safe  with  oil- 
immersion  lenses  ;  whereas  an  alcohol-balsam  mount  will  be 
dry  enough  in  a  couple  of  days. 


286  CHAPTER    XX. 

REHM  (Zeit.  f.  wiss.  Mik.,  ix,  1893,  p.  387)  recommends  a  solution  of 
1  part  of  colophoniuin  in  10  of  benzin;  and  later  writers  also  recommend  a 
similar  solution. 

444,  Venice  Turpentine  for  Mounting  (VOSSELER,  Zeit.  /.  wiss. 
Mik.,  vi,  3,  1889,,  p.  292,  et  seq.). — Commercial  Venice  turpen- 
tine is  mixed  in  a  tall  cylinder  glass,  with  an  equal  volume  of 
96  per  cent,  alcohol,  allowed  to  stand  in  a  warm  place   for 
three  or  four  weeks,  and  decanted.    It  is  stated  that  prepara- 
tions may  be  mounted  in  this  medium  without  previous  clear- 
ing with  essential  oils  or  the  like.      The  index  of  refraction 
being  lower  than  that  of  the  above-named  balsams,  delicate 
details  are  more   distinctly  brought  out.      Stains  keep  well, 
according  to  VOSSELER. 

MAYER  (Grundzuge,  p.  236)  notes  hereon  that  not  all  stains 
will  keep  well  in  it  011  account  of  the  alcohol  and  oil  of  tur- 
pentine in  it ;  hsemalum  stains  fade  rapidly  in  it.  He  con- 
siders it  a  very  valuable  medium  on  account  of  its  faculty  of 
supporting  a  notable  proportion  of  water  in  the  preparations. 
Celloidin  sections  can  be  mounted  direct  from  96  per  cent, 
alcohol ;  it  does  not  ca,use  turbidity  in  the  albumen  of  MAYER'S 
fixative  for  sections,  and  you  may  breathe  on  it  with  impunity 
whilst  mounting.  This  faculty  of  withstanding*  moisture 
makes  it  especially  valuable  at  the  seaside. 

This  medium  is  also  recommended  by  SUCHANNEK  (ibid., 
vii,  4,  1891,  p.  463).  He  advises  that  it  be  prepared  with 
equal  parts  of  Venice  turpentine  and  neutral  absolute  alcohol 
(obtained  by  treating  commercial  absolute  alcohol  with  cal- 
cined cupric  sulphate  and  quicklime).  The  mixture  should 
be  agitated  frequently  and  kept  in  a  tile  stove  for  a  day  or 
two  until  clear  and  sufficiently  inspissated. 

445,  Thickened  Oil  of  Turpentine  ("  Verhartzes  Terpentinol" 
of  German  writers)  has  been  used  as  a  mounting  medium  by 
some  workers.      It  is  prepared  by  exposing  rectified  oil  of 
turpentine  in  thin  layers  for  some  days  to  the  air.      All  that 
is  necessary  is  to  pour  some  oil  into  a  plate,  cover  it  lightly 
so  as  to  protect  it  from  dust  without  excluding  the  air,  and 
leave  it  until  it  has  attained  a  syrupy  consistency. 

446,  Cedar  Oil. — I  most  highly  recommend  this  oil,  both  as 
a  temporary  examination  medium  and  as  a  mounting  medium. 
See  §  438. 


EXAMINATION    AND    PRESERVATION   MEDIA.  287 

447.  Castor  Oil. — This  was  recommended  as  a  mounting  medium  for 
certain  delicate  tissues  (sections  of  eyes  of  Cephalopods)  by  GBENACHEK 
(Abhandl.  naturf.  Ges.  Halle- a.-S.,  Bd.  xvi ;    Zeit.  f.  wiss.  Mik.,  1885, 
p.  244).     This  was  with  the  idea  that  its  low  refractive  index  (11  =  T49, 
whilst  Canada  balsam  n  =  1'54)  would  give  a  useful  augmentation  of  visi- 
bility for  the  more  refractive  elements  of  the  tissues. 

With  the  objects  with  which  I  have  experimented  I  have  not  had  good 
results. 

448.  Photographic  Negative  Varnish  (for  mounting  large  sections 
without  cover-glasses).— See  WEIGEET,  Zeit.f.  wiss.  Mik.,  iv,  2, 1887,  p.  209. 

449.  Styrax  and  Liquidambar. — See  Journ.  Roy.  Mic.  Soc.,  1883, 
p.  741;  ib.,  1884,  pp.  318,  475,  655,  and  827;  and  the  places  there  quoted. 
Also  Bull.  Soc.  Beige  de  Mic.,  1884,  p.  178;    and  FOL,  Lehrb.,  p.  141. 
These  are  very  highly  refractive  media,  which  is  just  what  is  not  wanted  in 
general  in  histology. 

450.  Sandarac  (LAVDOWSKY,  from  Ref.  Handbook  Med.  Sci.,  Supp., 
p.  438). — Gum  sandarac  30  grs.,  absolute  alcohol  50  c.c.     This  may,  if 
desired,  be  diluted  with  an  equal  volume  of  absolute  alcohol,  and  used  for 
clearing  sections. 

450a.  Gum  Thus,  dissolved  in  xylol,  is  recommended  by  EISEN,  Zeit. 
f.  wiss.  Mik.,  xiv,  1897,  p.  201. 


CHAPTER  XXI. 

CEMENTS    AND  VAENI8HES. 

451.  Introduction. — Two,  or  at  most  three,  of   the   media 
given  below  will  certainly  be  found  sufficient  for  all  useful 
purposes.      For   many  years   I   have  used  only  one   cement 
(BELI/S).     I  recommend  this  as  a  cement  and  varnish  ;   gold 
size  may  be  found   useful  for  turning  cells  ;    and  MILLER'S 
caoutchouc  cement  may  be  kept  for  occasions  on  which  the 
utmost  solidity  is  required. 

Marine  glue  is  necessary  for  making  glass  cells. 

CARPENTER  lays  great  stress  on  the  principle  that  the 
cements  or  varnishes  used  for  fluid  mounts  should  always  be 
such  as  contain  no  mixture  of  solid  particles  •  he  has  always 
found  that  those  that  do,  although  they  might  stand  well 
for  a  few  weeks  or  months,  yet  always  become  porous  after 
a  greater  lapse  of  time,  allowing  the  evaporation  of  the 
liquid  and  the  admission  of  air.  All  fluid  mounts  should  be 
ringed  with  glycerin  jelly  before  applying  a  cement;  by  this 
means  all  danger  of  running  in  is  done  away  with.  See  §§ 
453  and  454. 

The  reader  who  requires  more  information  concerning 
microscopical  cements  and  varnishes  than  can  be  given  in 
this  chapter  may  consult  with  advantage  the  papers  of 
AUBERT,  The  Microscope ,  xi,  1891,  150,  and  Journ.  Roy.  Mic. 
Soc.,  1891,  p.  692  •  BECK,  The  Microscope,  xi,  1891,  pp.  338, 
368,  and  Journ.  Roy.  Mic.  Soc.,  1892,  p.  293;  BEHRENS' 
Tabellen  zum  Gebrauch  bei  mikroskopischen  Arbeiten  (Bruhn, 
Braunschweig,  1892)  ;  and  ROUSSELET,  "  On  some  Micro- 
Cements,"  Journ.  Quekett  Mic.  Club,  vii,  1898,  p.  93. 

452.  Comparative  Tenacity  of  Cements   (see  BEHRENS,  Zeit.  f. 
wiss.  Mik.,  ii,  1885,  p.  54,  and  AUBERT,  Amer.    Mon.   Mic. 


CEMENTS    AND    VARNISHES.  289 

Journ.,  1885,  p.  227;  Joui-n.  Roy.  Mic.  Soc.,  1886,  p.  173). 
— BEHRENS  gives  the  palm  to  amber  varnish  ;  AUBERT  places 
MILLER'S  caoutchouc  cement  at  the  head  of  the  list,  LOVETT'S 
cement  coming  halfway  down,  and  zinc  white  cement  at  the 
bottom,  with  less  than  one  quarter  the  tenacity  of  the  caout- 
chouc cement. 

453.  The  Paper  Cell  Method. — According  to  my  experience, 
the  best  way  to  make  a  fluid  mount  safe  is  the  following  : — 
By  means  of  two  punches  I  cut  out  rings  of  paper  of  about 
a  millimetre  in  breadth,  and  of  about  a  millimetre  smaller  in 
diameter  than  the  cover- glass.      Moisten  the  paper  ring  with 
mounting  fluid,  and  centre  it  on  the  slide.      Fill  the  cell  thus 
formed  with  mounting  fluid ;   arrange  the  object  in  it ;  put 
the  cover  on  ;   fill  the  annular  space  between  the  paper  and 
the  margin  of  the  cover  with  glycerin  jelly  (a  turn-table  may 
be  useful  for  this  operation) ;   and  as  soon  as  the  gelatin  has 
set  turn  a  ring  of  gold-size  on  it,  and  when  that  is  quite  dry, 
varnish  with  BELL'S  cement. 

For  greater  safety,  the  gelatin  may,  of  course,  be  treated 
with  bichromate,  according  to  MARSH'S  plan,  next  §. 

454.  Gelatin  Cement  (MARSH'S  Section-cutting,  2nd  ed.,  p. 
104). — Take  half  an  ounce  of  NELSON'S  opaque  gelatin,  soak 
well  in  water,  melt  in  the  usual  way,  stir  in  3  drops  of  crea- 
sote,  and  put  away  in  a  small  bottle.      It  is  used  warm. 

When  the  ring  of  gelatin  has  become  quite  set  and  dry, 
which  will  not  take  long,  it  may  be  painted  over  with  a 
solution  of  bichromate  of  potash  made  by  dissolving  10  grains 
of  the  salt  in  an  ounce  of  water.  This  should  be  done  in  the 
daytime,  as  the  action  of  daylight  is  necessary  to  enable  the 
bichromate  to  render  the  gelatin  insoluble  in  water.  The 
cover  may  then  be  finished  with  BELL'S  cement. 

This  process  is  particularly  adapted  for  glycerin  mounts. 

455.  ROUSSELET'S  Method  for  Aqueous  Mounts  (op.  ci£.,§451). 
— Close  the   mount  with  a  ring  of  -a,  mixture  of  two  parts 
of  a  solution  of  damar  in   benzol   and   one   part   gold-size. 
When  dry,  put  on  three  or  four  thin  coats  of  pure  gold-size 
at  intervals  of  twenty-four  hours,  and  finish  with  a  ring  of 
WARD'S  brown  cement. 

19 


290  CHAPTER  XXI. 

456.  WAED'S  Brown   Cement  is  a  shellac-alcohol  solution, 
made  by  E.  Ward,  Oxford  Road,  Manchester,  and  presumably 
now  obtainable   from  the  opticians.      Its  best  solvent,  Mr. 
ROUSSELET  writes   me,  is   a   mixture    of   wood-naphtha   and 
alcohol.      He  considers  it  the  best  shellac  varnish  he  has  met 
with,  better  than  BELL'S. 

457.  BELI/S  Cement. — Composition  unknown.      May  be  ob- 
tained from  the  opticians,  or  from  J.  Bell  &  Co.,  chemists, 
338,  Oxford  Street,  London. 

This  varnish  flows  easily  from  the  brush,  and  sets  quickly. 
For  glycerin  or  other  fluid  mounts  the  cover  should  be  ringed, 
as  above  described,  with  glycerin  jelly  before  applying  the 
varnish.  This  precaution  is  especially  necessary  with 
glycerin.  This  is  the  best  varnish  for  fluid  mounts  known 
to  me.  It  is  soluble  in  ether  or  chloroform.  It  is  not 
attacked  by  oil  of  cedar. 

458.  MILLEK'S  Caoutchouc  Cement. — Composition  unknown. 
May  be  obtained  from  the  opticians.      A  very  tenacious  and, 
which   is    frequently   an  important  point,  a   quickly  drying 
cement.      It  may  be  diluted  by  a  mixture  of  equal  parts  of 
chloroform  and  strong  alcohol    (see  ROUSSELET,  Journ.  Quek. 
Club,  v,  ii,  1895,  p.  8). 

459.  CLAKKE'S  Spirit-proof  Cement. — Mr.  CH.  ROUSSELET  has 
highly  recommended  this  to  me.      It  may  be  procured  from 
Mr.  J.  Bolton,  25,  Balshall  Heath  Road,  Birmingham. 

ROUSSELET  finds  it  the  best  he  has  tried  for  alcoholic 
liquids,  but  not  perfectly  proof  against  watery  media. 

460.  Asphalt  Varnish  (Bitume  de  Judee). — Unquestionably 
one  of  the  best  of  these  media,  either  as  a  cement  or  a  varnish, 
provided  it  be  procured  of  good  quality.      It  can  be  procured 
from  the  opticians  or  from  the  oil-shops. 

461.  Brunswick  Black. — See   previous    editions,   or  BEALE, 
How  to  Work,  etc.,  p.  49. 

462.  Gold-Size. — Receipts  for  preparing  it  may  be  found  in 
the  Micrographic  Diet,  or  in  COOLEY'S  Cyclopaedia  ;  but  it  is 


CEMENTS    AND   VARNISHES.  291 

certainly  best  to  obtain  it  from  the  opticians  or  oil-shops.  It 
is  soluble  in  oil  of  turpentine.  A  good  cement,  when  of  good 
quality,  and  very  useful  for  turning  cells. 

463.  Marine  Glue. — Found  in  commerce.      CARPENTER  says 
the  best  is  that  known  as  G  K  4. 

It  is  soluble  in  ether,  naphtha,  or  solution  of  potash.  Its 
use  is  for  attaching  glass  cells  to  slides,  and  for  all  cases  in 
which  it  is  desired  to  cement  glass  to  glass. 

Eeceipts  for  preparing  it  may  be  found  in  BEALE,  p.  40,  or 
in  COOLEY'S  Cyclopaedia. 

464.  Turpentine,   Venice  Turpentine    (CsoKOR,    Arch.   mik. 
Anat.}  xxi,  1882,  p.  853;  PARKER,  Amer.  Mon.   Mic.  Journ., 
ii,  1881,  pp.  229-30;  Journ.  Roy.  Mic.  Soc.  [N.  S.],  ii,  1882, 
p.    724). — Venice   turpentine    (Terebinthina  veneta)    is  the 
liquid  resinous  exudation  of  Abies  larix.      Parker  gives  the 
following  directions  : 

Dissolve  true  Venice  turpentine  in  enough  alcohol,  so  that 
after  solution  it  will  pass  readily  through  a  filter,  and,  after 
filtering,  place  in  an  evaporating  dish,  and  by  means  of  a 
sand-bath  evaporate  down  to  about  three  quarters  of  the 
quantity  originally  used.  After  it  has  evaporated  down  to 
about  that  much,  drop  some  of  the  mass  into  cold  water ;  if 
on  being  taken  out  of  the  water  it  is  hard  and  breaks  with 
a  vitreous  fracture  on  being  struck  with  the  point  of  a  knife, 
cease  evaporation  and  allow  to  cool. 

Or  (CSOKOR),  common  resinous  turpentine  of  commerce  is 
put  in  small  pieces  to  melt  over  a  water-bath,  then  poured 
into  a  suitable  vessel  and  allowed  to  cool.  It  should  form  a 
brittle,  dark  brown  mass,  not  yielding  to  the  pressure  of  a 
finger.  It  is  sometimes  useful,  in  order  to  attain  the  right 
degree  of  hardness  in  the  cold  mass,  to  add  a  little  resinous 
oil  of  turpentine  to  the  melted  mass,  and  then  to  evaporate 
for  several  hours  over  the  water- bath. 

This  cement  is  used  for  closing  glycerine  mounts;  it  is 
applied  in  the  following  manner : — Square  covers  are  used, 
and  superfluous  glycerin  is  cleaned  away  from  the  edges  in 
the  usual  way. 

The  cement  is  then  put  on  with  a  piece  of  wire  bent  at 
right  angles ;  the  short  arm  of  the  wire  should  be  just  the 


292  CHAPTER   XXI. 

length  of  the  side  of  the  cover-glass.  The  wire  is  heated  in 
a  spirit  lamp,  plunged  into  the  cement,  some  of  which  ad- 
heres to  it,  and  then  brought  down  flat  upon  the  slide  at  the 
margin  of  the  cover.  The  turpentine  distributes  itself  evenly 
along  the  side  of  the  cover,  and  hardens  immediately,  so  that 
the  slide  may  be  cleaned  as  soon  as  the  four  sides  are 
finished.  It  is  claimed  for  this  cement  that  it  is  perfectly 
secure,  very  handy,  and  never  runs  in.  The  cement  sets 
hard  in  a  few  seconds. 

465.  Colophonium   and  Wax   (KEONIG,  Arch.  f.  mik.  Anat.t 
1886,  p.  657;  Jour n.  Roy.  Mic.  Soc.,  1887,  p.  344).— Seven 
to  nine   parts   of   colophonium  are  added  piecemeal  to  two 
parts  of  melted  wax,  the  whole  filtered  and  left  to  cool.      For 
use,  the  mass  is  melted  by  placing  the  containing  vessel  in 
hot  water.      The  cement  is  not  attacked  by  water,  glycerin, 
or  caustic  potash. 

466.  APATHY'S   Cement  for  Glycerin  Mounts    (Zeit.  f.   wiss. 
Mik.,  vi,  2,  1889,  p.  171).— Equal  parts  of  hard  (60°  C.  melt- 
ing-point) paraffin  and  Canada  balsam.      Heat  together  in  a 
porcelain  capsule  until  the  mass  takes  on  a  golden  tint  and 
no   longer   emits  vapours   of  turpentine.      On  cooling,  this 
forms  a  hard  mass,  which  is  used  by  warming  and  applying* 
with  a  glass  rod  or  brass  spatula.      One  application  is  enough. 
The  cement  does  not  run  in,  and  never  cracks. 

467.  Paraffin. — Temporary    mounts    may   be    closed    with 
pure  paraffin,  by  applying  it  with  a  bent  wire,  as  described 
§  464. 

468.  Canada  Balsam,  or  Damar. — Cells  are  sometimes  made  with 
these.  They  are  elegant,  but  in  my  experience  are  not  reliable  for  per- 
manent mounts. 

469.  Amber  Varnish. — As  above  mentioned,  BEHRENS  finds 
this  cement  to  possess  an  extreme  tenacity.      That  used  by 
him  may  be  obtained  from  Grubler  &  Hollborn. 

470.  Amber  and  Copal  Varnish  (HEYDENBEICH,  Zeit.  f.  wiss. 
Mik.,  1885,  p.   338). — Extremely  complicated;  may  be  ob- 
tained from  Ludwig  Marx,  at  110,  Moskowskaja   Sastawa, 


CEMENTS    AND    VARNISHES.  293 

St.    Petersburg ;  or   79,  Gaden,  Vienna ;  or   1,   Komerthal, 
Mayence. 

471.  Shellac  Varnish  (BE ALE,  p.  28). — Shellac  should  be  broken  into 
small  pieces,  placed  in  a  bottle  with  spirit  of  wine,  and  frequently  shaken 
until  a  thick  solution  is  obtained.     The  Micro.  Dictionary  says  that  the 
addition  of  20  drops  of  castor  oil  to  the  ounce  is  an  improvement. 

Untrustworthy,  but  useful  for  protecting  balsam  mounts  from  the  action 
of  oil  of  cedar. 

472.  Sealing- Wax  Varnish  (Micro.  Diet., "  Cements  ").— Add  enough 
spirit  of  wine  to  cover  coarsely  powdered  sealing-wax,  and  digest  at  a  gentle 
heat.    This  should  only  be  used  as  a  varnish,  never  as  a  cement,  as  it  is  apt 
to  become  brittle  and  to  lose  its  hold  upon  glass  after  a  time. 

473.  Tolu  Balsam  Cement  (CARNOY'S  Biol.  Cell,  p.  129). 
Tolu  balsam        .          .          .          .2  parts. 
Canada  balsam  .          .          .          .      1  part. 
Saturated    solution   of    shellac    in 

chloroform      . ,        .          *          .2  parts. 
Add  enough  chloroform  to  bring  the  mixture  to  a  syrupy 
consistence.     CARNOY  finds  this  cement  superior  to  all  others. 

474.  Other  Cements  and  Varnishes.— See  previous  edition*. 


PART   II. 

SPECIAL   METHODS    AND   EXAMPLES. 


CHAPTER  XXII. 

INJECTION GELATIN  MASSES. 

475.  Introduction. — Injection  masses  are   composed   of   a 
coloured  substance,   technically  termed  the  colouring  mass, 
and  of  a  substance  with  which  that  is  combined,  technically 
termed  the  vehicle. 

The  following  formulae  are  grouped  mainly  according  to 
the  nature  of  the  vehicle.  The  chief  vehicles  are  gelatin  and 
glycerin. 

For  injections  made  for  the  study  of  the  angiology  of  Ver- 
tebrates, the  student  will  do  well  to  follow  the  masterly  prac- 
tice of  ROBIN  and  RANVIEK,  consulting  also,  if  necessary, 
the  excellent  instructions  given  in  BE  ALE'S  How  to  Work 
with  the  Microscope,  and  in  the  Lehrbuch  der  vergleichenden 
mikroskopischen  Anatomic  of  FOL.  For  injections  of  Inverte- 
brates (and,  indeed,  for  Vertebrates  if  it  is  desired  to  demon- 
strate the  minute  structure  of  environing  tissues  at  the  same 
time  as  the  distribution  of  vessels)  glycerin  masses  are 
generally  preferable  to  gelatin  masses ;  and  I  would  recom- 
mend as  particularly  convenient  the  Prussian  blue  glycerin 
masses  of  BE  ALE.  Glycerin  masses  have  the  great  advantage 
that  they  are  used  cold. 

All  formulae  which  only  give  opaque  masses,  or  are  only 
suitable  for  coarse  injections  for  naked-eye  study,  have  been 
suppressed. 

476.  Nitrite  of  Amyl  as  a  Vaso-dilator. — As  stated  above, 
glycerin  masses  are  certainly  very  convenient,  and  give  very 
good   results  from  the   scientific — not  from   the   aesthetic — 
point  of  view.      They  have  a  great  defect  for  the  injection 
of  fresh  specimens — that  is,  those  in  which  rigor  mortis  has 
not  set  in ;  they  stimulate  the  contraction  of  arteries.      In 


298  CHAPTER    XXII. 

these  cases  it  may  be  advisable  to  use  nitrite  of  amyl  as  a 
vaso-dilator.  The  animal  may  be  anaesthetised  with  a  mix- 
ture of  ether  and  nitrite  of  amyl,  and  finally  killed  with 
pure  nitrite.  Or,  after  killing  by  nitrite,  a  little  nitrite  of 
amyl  in  salt  solution  may  be  injected  before  the  injection 
mass  is  thrown  in.  In  any  case  it  is  advisable  to  add  a 
little  nitrite  to  the  mass  just  before  using.  The  relaxing 
power  is  very  great  (see  OVIATT  and  SARGENT,  in  St.  Louis 
Med.  Journ.,  1886,  p.  207  ;  and  Journ.  Roy.  Mic.  Soc.,  1887, 
p.  341). 

477.  Foi/s  Metagelatin  Vehicle  (Lehrb.,  p.  17).— The  opera- 
tion of  injecting  with  the  ordinary  gelatin  masses  is  greatly 
complicated  by  the  necessity  of  injecting  them  warm.  FOL 
proposes  to  employ  metagelatin  instead  of  gelatin. 

If  a  slight  proportion  of  ammonia  be  added  to  a  solution 
of  gelatin,  and  the  solution  be  heated  for  several  hours,  the 
solution  passes  into  the  state  of  metagelatin,  that  is-,  a  state 
in  which  it  no  longer  coagulates  on  cooling.  Colouring 
masses  may  be  added  to  this  vehicle,  which  may  also  be 
thinned  by  the  addition  of  weak  alcohol.  After  injection, 
the  preparations  are  thrown  into  strong  alcohol  or  chromic 
acid,  which  sets  the  mass. 


KOBIN'S  Masses. 

478.  ROBIN'S  Gelatin  Vehicle  (Traite,  p.  30). — Take  some 
good  gelatin,  soak  it  in  cold  water,  then  heat  in  water  over 
a  water-bath.  One  part  of  gelatin  should  be  taken  for 
every  7,  8,  9,  or  even  10  parts  of  water  ;  it  is  a  common 
error  to  employ  solutions  containing  too  much  gelatin.  The 
solution  is  now  to  be  combined  with  one  of  the  colouring 
masses  given  below. 

This  vehicle,  like  all  gelatin  masses,  is  liable  to  be  attacked 
by  mould  if  kept  long  ;  camphor  and  carbolic  acid  do  not 
suffice  to  preserve  it. 

Chloral  hydrate  added  to  the  mass  is  said  to  preserve  it 
(HOYER).  A  sufficient  dose,  at  least  2  per  cent.,  should  be 
employed  (see  below,  §  485). 


INJECTION GELATIN    MASSES.  299 

479.  ROBIN'S  Glycerin-gelatin  Vehicle  (Traite,  p.  32). — Dis- 
solve in  a  water-bath  50  grms.  of  gelatin  in  300  grms.  of 
water,   in  which   has  been   dissolved   some   arsenious   acid ; 
add  of  glycerin  150  grms.,  and  of  carbolic  acid  a  few  drops. 
Unlike  the  pure  gelatin  vehicles,  this  mass  does  keep  in- 
definitely. 

The  colouring  masses  recommended  for  combination  with 
the  vehicles  above  described  are  given  in  §§  480  to  483,  and 
§497. 

FBANKL  (Zeit.f.  wiss.  ZooL,  Ixiii,  1897,  p.  28)  prepares  a  similar  vehicle, 
and  adds  to  it  a  little  solution  of  corrosive  sublimate  and  a  crystal  of 
thymol. 

480.  ROBIN'S  Carmine  Colouring  Mass  (Traite,p.33). — Rub 
up  in  a  mortar  3  grms.  of  carmine  with  a  little  water  and 
enough  ammonia  to  dissolve  the  carmine.      Add  50  grms.  of 
glycerin,  and  filter. 

Prepare  50  grms.  of  acid  glycerin  (containing  5  grms.  of 
acetic  acid  for  every  50  grms.  of  glycerin),  and  add  it  by 
degrees  to  the  carmine-glycerin,  until  a  slightly  acid  reaction 
is  obtained  (as  tested  by  very  sensitive  blue  test-paper,  moist- 
ened and  held  over  the  mixture). 

One  part  of  this  mixture  is  to  be  added  to  3  or  4  parts  of 
the  gelatin  injection  vehicle  (ante,  §  478),  or  of  the  glycerin- 
gelatin  (§479). 

481.  Ferrocyanide  of  Copper  Colouring  Mass  (ibid.,  p.  34). 
—Take— 

(1)  Ferrocyanide.  of  potassium  (concentrated 

solution)  ...  .20  c.c. 

Glycerin    .  .  •  •  •  •   50    „ 

(2)  Sulphate  of  copper  (concentrated  solu- 

tion)      .  .  .  .  .  .   35    „ 

Glycerin    .  .  .  •  •  •   50    „ 

Mix  (1)  and  (2)  slowly,  with  agitation;  at  the  moment  of 
injecting  combine  with  3  volumes  of  vehicle. 

482.  Blue  Colouring  Mass  (Prussian  Blue)  (ibid.,  p.  35,  and 
2nd  ed.,  p.  1013). 


300  CHAPTER   XXII. 

Take— 

(A)  Ferrocyanide  of  potassium*  (sol.  sat.)  .   90  c.c. 
Glycerin    .           .           .           .           .  .   50    „ 

(B)  Liquid  perchloride  of  iron  at  30    .  3    „ 
Glycerin    .           .           .           .           .  .   50    „ 

Mix  slowly  and  combine  the  mixture  with  3  parts  of  vehicle. 
It  is  well  to  add  a  few  drops  of  HC1. 


Carmine-gelatin  Masses. 

483.  RANVIEK'S  Carmine-Gelatine  Mass  (Traite  technique,  p. 
116). — Take  5  grms.  Paris  gelatin,  soak  it  in  water  for  half 
an  hour,  or  until  quite  swollen  and  soft ;  wash  it ;  drain  it ; 
put  it  into  a  test-tube  and  melt  it,  in  the  water  it  has  absorbed, 
over  a  water-bath.  When  melted  add  slowly,  and  with  con- 
tinual agitation,  a  solution  of  carmine  in  ammonia,  prepared 
as  follows  : — 2\  grms.  of  carmine  are  rubbed  up  with  a  little 
water,  and  just  enough  ammonia,  added  drop  by  drop,  to 
dissolve  the  carmine  into  a  transparent  solution. 

When  the  carmine  has  been  added  to  the  gelatin,  you  will 
have  about  15  c.c.  of  ammoniacal  solution  of  carmine  in 
gelatin,  if  the  operations  have  been  properly  performed. 
This  solution  is  to  be  kept  warm  on  the  water-bath,  whilst 
you  proceed  to  neutralise  it  by  adding  cautiously,  drop  by 
drop,  with  continual  agitation,  a  solution  of  1  part  of  glacial 
acetic  acid  in  2  parts  of  water.  (When  the  mass  is  near 
neutrality,  dilute  the  acetic  acid  still  further.)  The  instant 
of  saturation  is  determined  by  the  smell  of  the  solution, 
which  gradually  changes  from  ammoniacal  to  sour.  As 
soon  as  the  sour  smell  is  perceived  the  liquid  must  be  exa- 
mined under  the  microscope.  If  it  contains  a  granular 
precipitate  of  carmine,  too  much  acid  has  been  added,  and 
it  must  be  thrown  away. 

KAN  VIE  B  states  that  this  is  the  only  way  to  attain  to  per- 
fect neutralisation.  Trust  must  not  be  put  in  formulae  that 
profess  to  indicate  the  proportions  of  ammonia  and  acetic 
acid  necessary  for  neutralisation,  on  account  of  the  varia- 
tion in  strength  of  the  solutions  of  ammonia  kept  in  labora- 

*  Erratim  "  Sulphocyanide  "  in'  1st  edition  of  ROBIN'S  Traite. 


INJECTION GELATIN    MASSES.  301 

tories,  and  also  because  it  often  happens  that  commercial 
gelatin  is  acid. 

The  mass  having  been  perfectly  neutralised  is  strained 
through  new  flannel. 

484.  How  to  Neutralise  a  Carmine  Mass  (ViLLE,  Gaz.  hebd.  d. 
Sci.  med.  de  Montpellier,  Fev.,  1882 ;   may  be  had  separately 
from  Delahaye  et  Lecrosnier,   Paris) . — VILLE  points  out  that 
when  carmine  is  treated  with  ammonia  a  certain  proportion  of 
the  ammonia  combines  with   the   carmine  to  form   a   trans- 
parent purple  compound,  and  the  rest  remains  in  excess.      It 
is  this  excess  that  it  is  required  to  neutralise  precisely,  not 
the  whole  of  the  ammonia  employed. 

As  to  the  acidity  accidentally  found  in  commercial  gelatin, 
that  source  of  error  is  easily  eliminated.  Instead  of  soaking 
the  gelatin  in  water,  it  should  be  placed  in  a  large  funnel 
with  a  narrow  neck,  or  better,  in  a  stopcock  funnel,  and  the 
whole  should  be  placed  under  a  tap,  and  a  stream  of  water 
arranged  in  such  a  manner  that  the  gelatin  be  constantly 
completely  immersed.  Washing  for  an  hour  or  so  in  this 
way  will  remove  all  traces  of  acids  mechanically  retained  in 
the  gelatin. 

As  to  the  neutralisation  of  the  colouring  mass,  VILLE  is  of 
opinion  that  the  sour  smell  cannot  be  safely  relied  on  in 
practice.  He  considers  it  greatly  preferable  to  employ 
exceedingly  delicate  dichroic  litmus  paper  (litmus  paper 
sensitised  so  as  to  be  capable  of  being  used  equally  for  the 
demonstration  of  acids  and  bases) .  Such  paper  is,  I  believe, 
now  found  in  commerce ;  for  directions  for  preparing  it  and 
for  preserving  ammonia  without  loss  of  strength,  and  other 
details,  see  previous  editions. 

485.  HOYKR'S  Carmine-Gelatin  Mass  (Biol.   Centralb.,   1882, 
p.  21). — Take  a  concentrated  gelatin  solution  and  add  to  it 
the  needful  quantity  of  neutral  carmine  staining  solution  (loc. 
cit.j  p.  17).      Digest  in  a  water-bath  until  the  dark  violet-red 
colour  begins  to  pass  into  a  bright  red  tint.      Then  add  5 — 10 
per  cent,  by  volumes  of  glycerin,  and  at  least  2  per  cent,  by 
weight  of  chloral,  in  a  concentrated  solution.      After  passing 
through  flannel  it  can  be  k«pt  in  an  open  vessel  under  a  bell- 
glass. 


302  CHAPTER    XXTI. 

486.  Foi/s  Carmine-Gelatin  Mass  (Zeit.  f.  wiss.  ZooL, 
xxxviii,  p.  492). 

This  can  be  kept  in  the  dry  state  for  an  indefinite  length 
of  time.  (FoL  finds  that  the  addition  of  chloral  hydrate  to 
wet  masses  is  not  an  efficient  preservative.) 

One  kilog.  of  Simeon's  photographic  gelatin*  is  soaked 
for  a  couple  of  hours,,  until  thoroughly  soft,  in  a  small 
quantity  of  water.  The  water  is  then  poured  off  and  the 
gelatin  melted  over  a  water-bath,  and  one  litre  of  concen- 
trated solution  of  carmine  in  ammonia  is  poured  in  with  con- 
tinual stirring.  (The  carmine  solution  is  prepared  by  diluting 
strong  solution  of  ammonia  with  three  or  four  parts  of  water 
and  adding  carmine  to  saturation ;  the  undissolved  excess  of 
carmine  is  removed  by  filtration  just  before  the  solution  is 
added  to  the  gelatin.) 

To  the  mixture  of  gelatin  and  carmine,  which  should  have 
a  strong  smell  of  ammonia,  sufficient  acetic  acid  is  added  to 
turn  the  dark  purple  colour  of  the  mixture  into  the  well- 
known  blood-red  hue.  Exact  neutralisation  is  not  necessary. 
'The  mass  is  set  aside  until  it  has  become  firm,  and  is  then 
cut  up  into  pieces,  which  are  tied  up  in  a  piece  of  tulle  or 
fine  netting.  By  means  of  energetic  compression  with  the 
hand  under  water  (it  must  be  acidulated  water,  O'l  per  cent, 
acetic  acid,  otherwise  the  carmine  will  wash  out ;  cf.  Journ. 
Roy.  Mic.  Soc.,  iv,  part  3,  1884,  p.  474)  the  mass  is  driven 
out  through  the  meshes  of  the  stuff  in  the  shape  of  fine 
strings,  which  are  washed  for  several  hours  in  a  sieve  placed 
in  running  water  in  order  to  free  them  from  any  excess  of 
acid  or  ammonia.  The  strings  are  then  again  melted,  and 
the  molten  mass  is  poured  on  to  large  sheets  of  parchment 
paper  soaked  with  paraffin,  and  the  sheets  are  hung  up  to 
dry  in  an  airy  place.  When  dry  the  gelatin  can  easily 
be  separated  from  the  sheets,  and  may  be  cut  into  long 
.strips  with  scissors  and  put  away,  protected  from  dust  and 
•damp,  until  wanted  for  use.  In  order  to  get  the  mass 
ready  for  use,  all  that  is  necessary  is  to  soak  the  strips  for 
:a  few  minutes  in  water  and  melt  them  over  a  water-bath. 

The  process  maybe  simplified,  without  giving  very  greatly 
inferior  results,  as  follows  (Lelirl).,  p.  13).  Gelatin  in  sheets 
is  macerated  for  two  days  in  the  above-described  carmine 
*  Doubtless  any  good  photographic  gelatin  will  do  as  well. 


INJECTION — GELATIN    MASSES.  303 

solution,  then  rinsed  and  put  for  a  few  hours  into  water 
acidulated  with  acetic  acid.  It  is  then  washed  on  a  sieve 
for  several  hours  in  running  water,  dried  on  parchment  paper, 
and  preserved  as  above. 

This  mass  is  very  well  spoken  of. 

487.  Other  Carmine  Gelatin  Masses. — THIEESCH'S,  see  Arch.f.  mik. 
Anat.,  1865,  p.  148.  GEBLACH'S,  see  EANVIEE,  Traite,  p.  118.  CARTEE'S, 
see  BEALE,  p.  113.  DAVIES,  see  his  Prep,  and  Mounting  of  Mic.  Objects, 
p.  138. 

Blue  Gelatin  Masses. 

488.  EOBIN'S  Prussian  Blue  Gelatin  Mass  (see  above,  §  482). 

489.  RAN  VIBE'S  Prussian  Blue  Gelatin  Mass  (Traite,  p.  119). 
— Twenty-five  parts  of  a  concentrated   aqueous   solution   of 
soluble   Prussian   blue   (prepared  as   directed  below)   mixed 
with  one  part  of  solid  gelatin. 

The  mixture  of  the  Prussian  blue  with  the  vehicle  is 
effected  in  the  following  manner  : 

Weigh  the  gelatin,  soak  it  in  water  for  half  an  hour  or  an 
hour,  wash  it,  and  melt  it  in  a  test-tube,  in  the  water  it  has 
absorbed,  by  heating  over  a  water-bath.  Put  the  solution 
of  Prussian  blue  into  another  test-tube,  and  heat  it  on  the 
same  water-bath  as  the  gelatin,  so  as  to  have  the  two  at  the 
same  temperature.  Pour  the  gelatin  gradually  into  the 
Prussian  blue  solution,  stirring  continually  with  a  glass  rod. 
Continue  stirring  until  the  disappearance  of  the  curdy  pre- 
cipitate that  forms  at  first.  (Some  gelatins  produce  a  per- 
sistent precipitate ;  these  must  be  rejected ;  but  it  must  be 
borne  in  mind  that  the  precipitate  that  invariably  forms  in 
even  the  best  gelatins  disappears  if  the  heating  be  continued.) 
As  soon  as  the  glass  rod  has  ceased  to  show  blue  granula- 
tions on  its  surface  on  being  withdrawn  from  the  liquid,  it 
may  be  concluded  that  the  Prussian  blue  is  completely  dis- 
solved. Filter  through  new  flannel,  and  keep  the  filtrate  at 
40  over  a  water-bath  until  injected. 

The  soluble  Prussian  blue  for  the  above  mass  is  prepared 
as  follows  : 

490.  Soluble  Prussian  Blue  for  Injection  Masses   (RANVIER, 
ibid.). — Make  a  concentrated  solution  of  sulphate  of  peroxide 


304  CHAPTER  XXII. 

of  iron  in  distilled  water,  and  pour  it  gradually  into  a  con- 
centrated solution  of  yellow  prussiate  of  potash.  There  is 
produced  a  precipitate  of  insoluble  Prussian  blue.  (An 
excess  of  prussiate  of  potash  ought  to  remain  in  the  liquid ; 
in  order  to  ascertain  whether  this  is  the  case  take  a  small 
quantity  of  the  liquid  and  observe  whether  a  drop  of  sulphate 
of  iron  still  precipitates  it.)  Filter  the  liquid  through  a  felt 
strainer,  underneath  which  is  arranged  a  paper  filter  in  a 
glass  funnel.  The  liquid  at  first  runs  clear  and  yellowish 
into  the  lower  funnel ;  distilled  water  is  then  poured  little  by 
little  on  to  the  strainer ;  gradually  the  liquid  issuing  from 
the  strainer  acquires  a  blue  tinge,  which,  however,  is  not 
visible  in  that  which  issues  from  the  lower  filter.  Distilled 
water  is  continually  added  to  the  strainer  for  some  days  until 
the  liquid  begins  to  run  off  blue  from  the  second  filter. 
The  Prussian  blue  has  now  become  soluble.  The  strainer 
is  turned  inside  out  and  agitated  in  distilled  water ;  the 
Prussian  blue  will  dissolve  if  the  quantity  of  water  be  suffi- 
cient. 

The  solution  may  now  be  injected  just  as  it  is,  or  it  may 
be  kept  in  bottles  till  wanted,  or  the  solution  may  be 
evaporated  in  a  stove,  and  the  solid  residuum  put  away  in 
bottle. 

For  injections,  if  a  simple  aqueous  solution  be  taken, 
it  should  be  saturated.  Such  a  mass  never  transudes 
through  the  walls  of  vessels.  Or  it  may  be  combined  with 
one  fourth  of  glycerin,  or  with  the  gelatin  vehicle  above 
described. 

491.  BRUCKE'S  Soluble  Berlin  Blue  (Arch.f.  mik.  Anat.,  1865, 
p.  87). 

Make  a  solution  of  f errocyanide  of  potassium  containing  217 
grammes  of  the  salt  to  1  litre  of  water. 

Make  a  solution  of  1  part  commercial  chloride  of  iron  in 
10  parts  water. 

Take  equal  volumes  of  each,  and  add  to  each  of  them 
twice  its  volume  of  a  cold  saturated  solution  of  sulphate  of 
soda.  Pour  the  chloride  solution  into  the  f  errocyanide  solu- 
tion, stirring  continually.  Wash  the  precipitate  on  a  filter 
until  soluble,  dry  it,  press  between  blotting-paper  in  a  press, 
break  the  mass  in  pieces,  and  dry  in  the  air. 


INJECTION GELATIN    MASSES.  305 

The  concentrated  solution  of  the  colouring  matter  is  to  be 
gelatinised  with  just  so  much  gelatin  that  the  mass  forms  a 
jelly  when  cold. 

For  another  method,  see  previous  editions. 

492.  THIEKSCH'S  Prussian  Blue  Gelatin  Mass  (Arch.  f.  mik. 
Anat.,  i,  1865,  p.  148). 

Take— 

(1)  A  solution  of  1  part  gelatin  in  2  parts  water. 

(2)  A  saturated  aqueous  solution  of  sulphate  of  iron. 

(3)  A  saturated  aqueous  solution  of   red  prussiate  of 

potash. 

(4)  A  saturated  aqueous  solution  of  oxalic  acid. 

Now  (A)  mix  12  c.c.  of  the  iron  solution  with  one  ounce  of 
the  gelatin  solution  at  the  temperature  of  25°  R. 

Then  (B)  mix,  at  the  same  temperature,  24  c.c.  of  the  prus- 
siate solution  with  two  ounces  of  the  gelatin  solution. 

(c)  To  the  latter  mixture  add  first  24  c.c.  of  the  oxalic  acid 
solution,  stir  well,  and  then  add  the  gelatin  and  iron  mixture 
(A).  Stir  continually,  keeping  the  temperature  at  from  20° 
to  25°  R.,  until  the  whole  of  the  Prussian  blue  is  precipitated. 
Finally,  heat  over  a  water-bath  to  about  70°  R.  and  filter 
through  flannel. 

493.  FOL'S  Berlin  Blue  Gelatin  Mass  (Zeit.  f.  wiss.  Zool.,  xxxviii, 
1883,  p.  494).— A  modification  of  THIEESCH'S  formula,  last  §,  the  mass  being 
made  into  strings  and  dried,  as  with  the  carmine  mass,  §  486.     See  pre- 
vious editions. 

494.  Other  Blue  Gelatin  Masses.  -HOYEB'S,  Arch.  f.  mik.  Anat., 
1876,  p.  649;  GUIGNET'S,  Journ.de  Microgr.,  1889,  p.  94;  Joum.  Roy. 
Mic.  Soc.,  1889,  p.  463 ;  or  previous  editions  of  this  work. 

495.  HOYEE'S  Silver  Nitrate  Yellow  Gelatin  Mass  (Biol.  Gen- 
tralbl.,  ii,  1882,  pp.  19,  22;  Journ.  Roy.  Mic.  Soc.  [N.  S.],  iii, 
1883,  p.  142). — "  A  concentrated  solution  of  gelatin  is  mixed 
with  an  equal  volume  of  a  4  per  cent,  solution  of  nitrate  of 
silver  and  warmed.  To  this  is  added  a  very  small  quantity 
of  an  aqueous  solution  of  pyrogallic  acid,  which  reduces  the 
silver  in  a  few  seconds ;  chloral  and  glycerin  are  added  as 
before"  (No.  485). 

20 


306  CHAPTER   XXII. 

This  mass  is  yellow  in  the  capillaries  and  brown  in  the 
larger  vessels. 

496.  KANVIER'S  Gelatin  Mass  for  Impregnation  (Traite, 
p.  123). — Concentrated  solution  of  gelatin,  2,  3,  or  4  parts; 
1  per  cent,  nitrate  of  silver  solution,  1  part. 

497.  Other  Colours.— HOYEE'S  Green  (Biol.  Centralb.,  ii,  1882,  p.  19). 
Made  by  mixing  a  blue  mass  and  a  yellow  mass.  THIEBSCH'S  Green  (Arch. 
f.  mik.  Anat.,  1865,  p.  149).  EOBIN'S  SCHEELE'S  Green  (ROBIN,  Traite, 
p.  37).  HABTING'S  "White  (see  FEEY,  Le  Microscope,  p.  190).  FEET'S 
White  (ibid.).  TEICHMANN'S  White  (ibid.,  p.  191).  FOL'S  Brown  (Z&it. 
f.  wiss.  Zool.,  xxxviii,  1883,  p.  494).  MILLEE'S  Purple  (see  Amer.  Mon. 
Mic.  Journ.,  1888,  p.  50;  Journ.  Roy.  Hie.  Soc.,  1888,  p.  518;  Zeit.  f. 
wiss.  Mik.,  v,  3,  1888,  p.  361).  FOL'S  Lead  Chromate  (Lehrb.,  p.  15). 
EOBIN'S  Cadmium  (his  Traite,  p.  36).  THIEBSCH'S  Lead  Chromate 
(Arch.  f.  mik.  Anat.,  1865,  p.  149).  HOYEE'S  Lead  Chromate  (ibid., 
1867,  p.  136) ;  or,  for  any  of  these,  previous  editions. 


CHAPTER    XXIII. 

INJECTIONS — OTHER  MASSES    (COLD). 

498.  JOSEPH'S  White-of-Egg  Injection  Mass  (Carmine)  (Ber. 
naturw.  Sect.  Schles.  Ges.,  1879,  pp.  36 — 40;  Journ.  Roy.  Mic. 
Soc.  [N.  S.],  ii,  1882,  p.  274).— "Filtered  white-of-egg, 
diluted  with  1  to  5  per  cent,  of  carmine  solution.  .  .  .  This 
mass  remains  liquid  when  cold ;  it  coagulates  when  immersed 
in  dilute  nitric  acid,  chromic  or  osmic  acid,  remains  trans- 
parent, and  is  sufficiently  indifferent  to  reagents." 

For  Invertebrates. 

499.  BJELOUSSOW'S  Gum  Arabic  Mass  (Arch.  f.  Auat.  u. 
Phys.,  1885,  p.  379) . — Make  a  syrupy  solution  of  gum  arabic 
and  a  saturated  solution  of  borax  in  water.  Mix  the  solu- 
tions in  such  proportions  as  to  have  in  the  mixture  I  part  of 
borax  to  2  of  gum  arabic.  Rub  up  the  transparent,  almost 
insoluble  mass  with  distilled  water,  added  little  by  little,  then 
force  it  through  a  fine-grained  cloth.  Repeat  these  opera- 
tions until  there  is  obtained  a  mass  that  is  free  from  suspended 
gelatinous  clots.  (If  the  operation  has  been  successful,  the 
mass  should  coagulate  in  the  presence  of  alcohol,  undergoing 
at  the  same  time  a  dilatation  to  twice  its  original  volume.) 

The  vehicle  thus  prepared  may  be  combined  with  any 
colouring  mass  except  cadmium  and  cobalt. 

After  injection  the  preparation  is  thrown  into  alcohol,  and 
the  mass  sets  immediately,  swelling  up  as  above  described, 
and  consequently  showing  vessels  largely  distended. 

Cold-blooded  animals  may  be  injected  whilst  alive  with 
this  mass.  It  does  not  flow  out  of  cut  vessels.  Injections 
keep  well  in  alcohol.  Glycerin  may  be  used  for  making 
them  transparent. 

If  it  be  desired  to  remove  the  mass  from  any  part  of  a 


308  CHAPTER   XXIII. 

preparation,  this  is  easily  done  with  dilute  acetic  acid,  which 
dissolves  it. 


Glycerin  Masses.* 

500.  BEALE'S  Carmine  Glycerin  Mass  (How  to   Work,  etc., 
p.  95) . — Five  grains  of  carmine  are  dissolved  in  a  little  water 
with  the  aid  of  about  five  drops  of  ammonia,  and  added  to 
half   an   ounce   of    glycerin.      Then   add  half    an   ounce    of 
glycerin  with  eight   or  ten  drops  of  acetic  or  hydrochloric 
acid,  gradually,  with  agitation.    Test  with  blue  litmus  paper, 
and  if  necessary  add  more  acid  till  the  reaction  is  decidedly 
acid.      Then  add  half  an  ounce  of  glycerin,  two  drachms  of 
alcohol,  and  six  drachms  of  water.    I  have  found  this  useful, 
but  not  so  good  as  the  Prussian  blue  injections. 

501.  BEALE'S  Prussian  Blue  (How  to  Work,  etc.,  p.  93). 
Common  glycerin       .  .         ' .        1  ounce. 
Spirits  of  wine            .           -.  ,'       .        1       „ 
Ferrocyanide  of  potassium.       \  .,'    12  grains. 
Tincture  of  perchloride  of  iron    .        1  drachm. 
Water     .           .           .          ,    ,       .        4  ounces. 

Dissolve  the  ferrocyanide  in  one  ounce  of  the  water  and 
glycerin,  and  add  the  tincture  of  iron  to  another  ounce. 
These  solutions  should  be  mixed  together  very  gradually,  and 
well  shaken  in  a  bottle,  the  iron  being  added  to  the  solution  of 
the  ferrocyanide  of  potassium.  Next,  the  spirit  and  the  rest 
of  water  are  to  be  added  very  gradually,  the  mixture  being 
constantly  shaken. 

Injected  specimens  should  be  preserved  in  acidulated 
glycerin  (e.g.  with  1  per  cent,  acetic  acid),  otherwise  the 
colour  may  fade. 

502.  BEALE'S  Acid  Prussian  Blue  (ibid.,  p.  296). 
Price's  glycerine        ...        2  fluid  ounces. 

.     Tinct.  of  sesquichloride  of  iron  .  10  drops. 

Ferrocyanide  of  potassium.  .  3  grains. 

Strong  hydrochloric  acid   .  .  3  drops. 

Water     .          .       .    .          .  .  1  ounce. 

*  See  the  remarks  on  Glycerin  Masses,  §  476. 


INJECTIONS OTHKR    MASSES    (<X>I.D).  309 

Proceed  as  directed  above,  dissolving  the  ferrocyanide  in 
one  half  of  the  glycerin,  the  iron  in  the  other,  and  adding 
the  latter  drop  by  drop  to  the  former.  Finally  add  the  water 
and  HC1.  Two  drachms  of  alcohol  may  be  added  to  the 
whole  if  desired. 

I  consider  this  a  most  admirable  formula.  The  mass  runs 
well,  and  has  not  so  much  tendency  to  exude  from  cut  capil- 
laries as  might  be  supposed.  Unfortunately  it  is  a  rather 
expensive  preparation. 

503.  RAXVIEE'S  Prussian  Blue  Glycerin  Mass  (Traite,  p.  120). — 
Consists  of  the  Prussian  blue  fluid,  §  490,  mixed  with  one  fourth  of  gly- 


504.  Other  Colours. — Any  of  the  colouring  masses,  §§  480  to  482,  or 
other  suitable  colouring  masses,  combined  with  glycerin,  either  dilute  or 
pure. 

505.  Gamboge  Glycerin  (HARTING,  Das  Mikroskop,  1866,  2 
Theil,  p.  124). — Gamboge  rubbed  up  with  water  and  added 
to  glycerin  ;  or  a  saturated  alcoholic  solution  of  gamboge 
added  to  a  mixture  of  equal  parts  of  glycerin  and  water. 
Any  excess  of  alcohol  may  be  got  rid  of  by  allowing  the 
mass  to  stand  for  twenty-four  hours. 


Aqiieous  Masses. 

506.  RANVIER'S  Prussian  Blue  Aqueous  Mass  (Traite,  p.  120). 
— The    soluble   Prussian   blue,  §  490,  injected  without   any 
vehicle.      It  does  not  extra vasate. 

507.  MULLER'S  Berlin  Blue    (Arch.  f.  mik.  Anat.,   1865,  p. 
150). — Precipitate  a  concentrated  solution  of  Berlin  blue  by 
means  of  4  to  1  volume  of  90  per  cent,  alcohol. 

The  precipitate  is  very  finely  divided ;   and  the  fluid  may 
be  injected  at  once. 

508.  MAYER'S  Berlin  Blue  (Mitth.  Zool.  Stat.  Neapel,  1888, 
p.  307). — A  solution  of  10  c.c.  of  tincture  of  perchloride  of 
iron  in  500  c.c.  of  water  is  added  to  a  solution  of  20  gr.  of 
yellow  prussiate  of  potash  in  500  c.c.  of  water,  allowed  to 


310  CHAPTBE   XXIII. 

stand  for  twelve  hours,,  decanted,,  the  deposit  washed  with 
distilled  water  on  a  filter  until  the  washings  come  through 
dark  blue  (one  to  two  days),  and  the  blue  dissolved  in  about 
a  litre  of  water. 

509.  EMEKY'S  Aqueous  Carmine  (ibid.,  1881,  p.  21).— To  a  10  per 
cent,  ammoniacal  solution  of  carmine  is  added  acetic  acid,  with  continual 
stirring,  until  the  colour  of  the  solution  changes  to  blood-red  through 
incipient  precipitation  of  the  carmine.     The  supernatant  clear  solution  is 
poured  off,  and  injected  cold  without  further  preparation.     The  injected 
organs  are  thrown  at  once  into  strong  alcohol  to  fix  the  carmine.     For 
injection  of  fishes. 

510,  TAGUCHI'S  Indian  Ink    (Arch.  f.  mik.  Anat.,  1888,  p. 
565  ;  Zeit.  f.  wiss.  Mik.,  1888,  p.  503). — Chinese  or  (better) 
Japanese  ink  well  rubbed  up  on  a  bone  until  a  fluid  is  ob- 
tained that  does  not  run  when  dropped  on  thin  blotting-paper, 
nor   form   a   grey    ring    round   the   drop.      Inject  until   the 
preparation  appears  quite  black,  and    throw    it    into    some 
hardening  liquid  (not  pure  water). 

I  believe  this  will  be  found  useful  for  work  amongst 
Invertebrates,  as  well  as  for  lymphatics,  juice  canals,  and  the 
like. 

Celloidin  Masses. 

511.  SCHIEFFEEDECZEB'S    Celloidin    Masses  (Arch.  Anat.  v.  Phys., 
1882  [Anat.  Abth.'],  p.  201).     (For  Corrosion  preparations  ) — See  previous 
editions  of  this  work,  or  WHITMAN'S  Methods  in  Microscopical  Anatomy. 

512.  HOCHSTETTER'S  Modification  of  SCHIEFFEBRECKER'S  Mass  (Anat. 
Anz.,  1886,  p.  51 ;  Journ.  Roy.  Mic.  Soc.,  1888,  p.  159). 


Other  Masses. 

513.  BUDGE'S  Asphaltum  Mass.— See  Arch.  f.  mik.  Anat.,  xiv,  1877, 
p.  70,  or  previous  editions. 

514.  HOYEB'S  Shellac  Mass  (Arch.f.  mik.  Anat.,  1876,  p.  645).— For 
this  and  that  of  BELLABMINOW  (Anat.  Anz.,  1888,  p.  650;  Journ.  Roy. 
Mic.  Soc.,  1889,  p.  150),  see  previous  editions. 

515.  HOYEB'S  Oil-colour  Masses  (Internat.  Monatsschr.f.  Anat..  1887, 
p.  341 ;  see  also  Zeit.  f.iviss.  Mik.,  1888,  p.  80,  and  Journ.  Roy.  Mic.  Soc., 


INJECTIONS OTHER   MASSES    (COLD).  311 

1888,  p.  848).  PANSCH'S  Starch  Mass  (see  Arch.f.  Anat.  u.  Entw.,  1877, 
p.  480;  1880,  pp.  232,  371 ;  1881,  p.  76;  1882,  p.  60;  1883,  p.  265  ;  and  a 
modification  of  the  same  by  GAGE,  Amer.  Hon.  Mic.  Joum.,  1888,  p.  195; 
and  Journ.  Roy.  Mic.  Soc.,  1888,  p.  1056).  TEICHMANN'S  Linseed-Oil 
Masses  (see  S.  B.  Math.  Kl.  Krakau  Akad.,  vii,  pp.  108,  158 ;  Joum. 
Roy.  Mic.  Soc.,  1882,  pp.  125  and  716,  and  1895,  p.  704). 

516.  Natural  Injections  (Rosm,  Traite,  p.  6). — To  preserve 
these  throw  the  organs  into  a  liquid  composed  of  10  parts  of 
tincture  of  per  chloride  of  iron  and  100  parts  of  water. 

RETTEEER  and  ZELLNEB  use  solution  of  Miiller,  see  Joum*. 
Eoy.  Mic.  Soc.,  1894,  p.  641. 


CHAPTER  XXIV. 

MACERATION    AND   DIGESTION. 
Maceration. 

517.  Methods  of  Dissociation, — It  is  sometimes  necessary, 
in  order  to  obtain  a  complete  knowledge  of  the  forms  of  the 
elements  of  a  tissue,  that  the  elements  be  artificially  separated 
from  their  place  in  the  tissue  and  separately  studied  after 
they  have  been  isolated  both  from  neighbouring  elements  and 
from  any  interstitial  cement-substances  that  may  be  present 
in  the  tissue.  Simple  teasing  with  needles  is  often  insufficient, 
as  the  cement -substances  are  frequently  tougher  than  the 
elements  themselves,  so  that  the  latter  are  torn  and  destroyed 
in  the  process.  In  this  case  recourse  must  be  had  to  macera- 
tion processes,  by  which  is  meant  treatment  with  media  which 
have  the  property  of  dissolving,  or  at  least  softening,  the 
cement -sub  stances  or  the  elements  of  the  tissue  that  it  is  not 
wished  to  study,  whilst  preserving  the  forms  of  those  it  is 
desired  to  isolate.  When  this  softening  has  been  effected, 
the  isolation  is  completed  by  teasing,  or  by  agitation  with 
liquid  in  a  test-tube,  or  by  the  method  of  tapping,  which 
last  gives  in  many  cases  (many  epithelia,  for  instance)  admir- 
able results  which  could  not  be  attained  in  any  other  way. 
The  macerated  tissue  is  placed  on  a  slide  and  covered  with 
a  thin  glass  cover  supported  at  the  corners  on  four  little  feet 
made  of  pellets  of  soft  wax.  By  tapping  the  cover  with  a 
needle  it  is  now  gradually  pressed  down,  whilst  at  the  same 
time  the  cells  of  the  tissue  are  segregated  by  the  repeated 
shocks.  When  the  segregation  has  proceeded  far  enough, 
mounting  medium  may  be  added,  and  the  mount  closed. 

The  student  will  do  well  not  to  neglect  this  simple  method. 

A  good  material  for  making  wax  feet  is  obtained  (YOSSELER, 


MACERATION    AND    DIGESTION.  313 

Zeit.  f.  wiss.  Mik.,  vii,  4,  1891,  p.  461)  by  melting  white  wax 
and  stirring  into  it  one  half  to  two  thirds  of  Venice  turpen- 
tine. Care  must  be  taken  if  the  operation  be  performed  over 
a  naked  flame,  as  the  turpentine  vapours  are  inflammable. 

518.  Iodised  Serum  (§  392). — The  manner  of  employing  it 
for  maceration  is  as  follows  : — A  piece  of  tissue  smaller  than 
a  pea  must  be  taken,  and  placed  in  4  or  5  c.c.  of  weakly 
iodised  serum    in    a    well-closed    vessel.      After    one    day's 
soaking    the    maceration    is    generally    sufficient,    and    the 
preparation  may  be  completed  by  teasing  or  pressing  out, 
as  indicated  above ;  if  not,  the  soaking  must  be  continued, 
fresh  iodine  being  added  as  often  as  the  serum  becomes  pale 
by  the  absorption  of  the  iodine  by  the  tissues.      By  taking 
this  precaution  the  maceration  may  be  prolonged  for  several 
weeks. 

These  methods  are  intended  to  be  applied  to  the  prepara- 
tion of  fresh  tissues,  the  iodine  playing  the  part  of  a  fixing 
agent  with  regard  to  protoplasm,  which  it  slightly  hardens. 

519.  Artificial  Iodised  Serum   (§   394). — RANVIER  has  been 
unable  to  obtain  good  results,  for  purposes  of  maceration, 
with  it. 

520.  Alcohol — RANVIER  employs  one  third  alcohol  (1  part 
of  90  per  cent,  alcohol  to  2  parts  of  water).      Epithelia  will 
macerate  well  in  this  in  twenty-four  hours.      RANVIER  finds 
that    this    mixture    macerates    more    rapidly    than    iodised 
serum. 

Other  strengths  of  alcohol  may  be  used,  either  stronger 
(equal  parts  of  alcohol  and  water)  or  weaker  (J  alcohol,  for 
isolation  of  the  nerve-fibres  of  the  retina,  for  instance — 
THIN). 

All  observers  are  agreed  that  one  third  alcohol  is  a  mace- 
rating medium  of  the  highest  order. 

521.  Salt  Solution. — 10  per  cent,  solution  of  sodium  chloride 
is  a  well-known  and  valuable  macerating  medium. 

522.  MOLESCHOTT   and  Piso  BORME'S  Sodium  Chloride  and 
Alcohol  (MOLESCHOTT'S  Untersuchimgen  zur  Naturlehre,  xi,  pp. 


314  CHAPTER   XXIV. 

99 — 107;  RANVIER,  Traite,  p.  242). — 10  per   cent,  solution 
of  sodium  chloride,  5  volumes  ;  absolute  alcohol,  1  volume. 

For  vibratile    epithelium   KANVIER  finds   the   mixture   in- 
ferior to  one  third  alcohol. 

523.  Formaldehyde. — G-AGE  recommends  the   addition  of  2 
parts  of  formalin  (40  per  cent,  solution  of  formaldehyde)  to 
1000  parts  of  normal  salt  solution.      The  mixture  acts  quickly, 
and  yet  retards  deterioration  for  some   time    (quoted  from 
FISH,  Proc.  Am.  Mic.  Soc.,  xvii,  1895,  p.  328). 

524.  Chloral  Hydrate.— In  not  too  strong  solution, from 2  to 
5  per  cent.,  for  instance,  chloral  hydrate  is  a  mild  macerating 
agent  that  admirably  preserves  delicate  elements.      LAVDOW- 
SKY  (Arch.f.  mik.  Anat.,  1876,  p.  359)  recommends  it  greatly 
for    salivary    glands.       HICKSON    (Quart.    Journ.    Mic.    Sci., 
1885,  p.  244)  recommends  it  for  the   study  of  the  retina  of 
Arthropods. 

525.  Caustic  Potash,  Caustic  Soda. — These  solutions  must  be 
employed  strong,  35  to  50  per  cent.   (MOLESCHOTT)  ;  so  em- 
ployed they  do  not  greatly  alter  the  forms  of  cells,  whilst 
weak  solutions  destroy  all   the   elements.      (Weak  solutions 
may,  however,  be  employed  for  dissociating  the  cells  of  epi- 
dermis, hairs,  and  nails.)      The  strong  solutions  may  be  em- 
ployed by  simply  treating  the  tissues  with  them  on  the  slide. 
To    make    permanent    preparations,    the    alkali    should    be 
neutralised  by  adding  acetic  acid,  which  forms  with  caustic 
potash  acetate   of   potash,  a  well-known  mounting   medium 
(see  BEHRENS,  KOSSEL,  and  SCHIEFFERDECKER,  Das  MilcrosJcop, 
i,  1889,  p.  156).      See  also  G-AGE,  Proc.  Amer.  Soc.  of  Micro- 
scopists,  1889,   p.   35  ;    Zeit.  f.   wiss.  Mik.,  vii,  3,    1890,  p. 
349. 

526.  Sulphocyanides  of  Ammonium  and  Potassium  (STIRLING, 
Journ.  Anat.  and  Phys.,  xvii,  1883,  p.  208). — 10  per  cent, 
solution  of  either  of  these  salts,  for  epithelium.      Macerate 
small  pieces  for  twenty- four  to  forty-eight  hours. 

527.  SOULIER'S  Sulphocyanide  Mixtures  (Travaux  de  VInst. 
Zool.  de  Montpdlier,  Nouv.  Ser.,  2,  1891,   p.  171). — SODLIER 


MACERATION    AND    DIGESTION.  315 

has  found  that  STIRLING'S  solution  greatly  deteriorates  cellular 
elements,  but  that  good  results  are  obtained  by  combining  it 
with  a  fixing  agent. 

The  best  results  were  obtained  with  a  2  per  cent,  solution 
of  sulphocyanide  combined  with  liquid  of  RIPART  and  PETIT. 

SOULIER  also  obtained  good  results  by  combining  liquid  of 
RIPART  and  PETIT  with  artificial  serum  of  KRONECKER  instead 
of  sulphocyanide,  or  with  pepsin,  eau  de  Javelle,  10  per  cent, 
sulphate  of  soda,  or  1*5  per  cent,  solution  of  caustic  soda. 

And  he  further  found  that  good  results  are  obtained  by 
combining  solutions  of  chloride  of  sodium,  or  solutions  of 
caustic  potash  or  soda,  with  any  of  the  usual  fixing  agents. 

528.  Saliva,  Artificial  (for  embryology  of  nerve  and  muscle) 
(CALBERLA,  Arch.  f.  mile.  Anat.,  xi,  1875,  p.  449). 

Second  formula  (the  first  is  suppressed)  : 

Potassium  chloride          .          ,          .  .  0*4 

Sodium  chloride   .  .  .  .  0'3 

Phosphate  of  soda          .          .  .  .  0*2 

Calcium  chloride  .  .          .  .  0*2 

1-1 
These  are  dissolved  in  100  parts  of  water,  saturated  with 

carbonic  acid,  and  the  solution  combined  with  half  a  volume 

of  MULLER'S  solution  and  a  volume  of  water. 

The  MULLER'S  solution  may  be  replaced  by  a  2£  per  cent. 

solution  of   chromate  of   ammonia.      The  best   results   were 

obtained   when   the  solutions   were  saturated  with  the  CO2 

just  before  using. 

529.  LANDOIS'S  Solution  (Arch.f.  mik.  Anat.,  1885,  p.  445). 
Saturated  sol.  of  neutral  chromate  of  ammonia  5  parts. 
Saturated  sol.  of  phosphate  of  potash       v          .          5     „ 
Saturated  sol.  of  sulphate  of  soda    .           .           .          5      „ 
Distilled  water      .          .          .•  .       .           .           .      100     „ 

Small  pieces  of  tissue  are  macerated  for  one  to  three,  or 
even  four  to  five  days,  in  the  liquid,  then  brought  for  twenty- 
four  hours  into  ammonia  carmine  diluted  with  one  volume  of 
the  macerating  liquid. 

GIERKK  particularly  recommends  this  liquid  for  all  sorts  of 
macerations,  but  especially  for  the  central  nervous  system,  for 


316  CHAPTER   XXIV. 

which  he  finds  it  superior  to  all  other  agents.  It  is  also  re- 
commended for  the  same  purpose  by  NAN  SEN  (v.  Zeit.  f.  iviss. 
Mik.,  v,  2,  1888,  p.  242). 

530.  Permanganate  of  Potash.. — Has  an  action  similar  to  that 
of  osmic  acid,  but  more  energetic.      Is  recommended,  either 
alone  or  combined  with  alum,  as  the  best  dissociating  agent 
for  the  fibres  of  the  cornea  (ROLLETT,  Strieker's  Handbuch, 
p.  1108). 

531.  Chromic  Acid. — Generally  employed  of  a  strength  of 
about  0*02  per  cent.      Specially  useful  for  nerve  tissues  and 
smooth  muscle.      Twenty-four  hours'  maceration  will  suffice 
for  nerve  tissue.      About  10  c.c.  of   the   solution  should  be 
taken  for  a  cube  of  5  mm.  of  the  tissue  (KANVIER). 

532.  Bichromate  of  Potash. — 0'2  per  cent. 

EISIG  (Fauna  u.  Flora  Golf.  Neapel,  16  Monog.,1887,  p.  297) 
macerates  Capitellidae  in  0'5  to  1  per  cent,  solution  for  months 
or  years,  a  little  thymol  being  added  against  mould. 

533.  Muller's  Solution. — Diluted  to  same  strength. 

534.  Miiller's  Solution  and  Saliva  (see  above,  §  528). 

535.  BROCK'S   Medium   (for  nervous    system   of   Mollusca, 
Intern.  Monatssch.  f.  Anat.,  i,  1884,  p.  349). — Equal  parts  of 
10  per  cent,  solution  of  bichromate  of   potash  and  visceral 
fluid  of  the  animal. 

536.  MOBIUS'S  Media  (Morph.  Jahrb.,  xii,  1887,  p.  174). 

1.  One  part  of  sea  water  with  4  to  6  parts  of  0*5  per  cent, 
solution  of  bichromate  of  potash. 

2.  0'25  per  cent,  chromic  acid,  0*1  per  cent,  osmic  acid, 
O'l  per  cent,  acetic  acid,  dissolved  in  sea  water.      For  Lamelli- 
branchiata.      Macerate  for  several  days. 

537.  GAGE'S  Picric  Alcohol  (Proc.  Amer.  Soc.  of  Microscopists, 
1890,  p.  120;  Zeit.}.  wiss.  Mik.,  ix,  1,  1892,  pp.  87,  88).— 95 
per   cent,   alcohol,   250   parts;    water,   750;   picric    acid,    1. 


MACERATION    AND    DIGESTION.  317 

Eecommended  especially  for  epithelia  and  muscle.      A  few 
hours'  maceration  is  generally  sufficient. 

538.  Osmic   and   Acetic   Acid  (the  HERTWIGS,  Da*  Nerven- 
system  u.  die  Sinnesorgane  der  Medusen,  Leipzig,  1878,  and 
Jen.  Zeitschr.,  xiii,    1879,  p.  457 ;  Journ.  Roy.  Mic.  Soc.,  iii, 
1880,  p.  441,  and  [N.  S.]  iii,  1883,  p.  732). 

0'05  per  cent,  osmic  acid        .  .      1  part. 

0*2  „  acetic  acid  .  .  1  „ 
Medusae  are  to  be  treated  with  this  mixture  for  two  or 
three  minutes,  according  to  size,  and  then  washed  in  repeated 
changes  of  O'l  per  cent,  acetic  acid  until  all  traces  of  free 
osmic  acid  are  removed;  they  then  remain  for  a  day  in  0*1 
per  cent,  acetic  acid,  are  washed  in  water,  stained  in  BE  ALE'S 
carmine,  and  preserved  in  glycerin. 

For  Actiniae  the  osmic  acid  is  taken  weaker,  0'04  per  cent. ; 
both  the  solutions  are  made  with  sea  water ;  and  the  washing 
out  is  done  with  0'2  per  cent,  acetic  acid.  If  the  maceration 
is  complete,  stain  with  picro-carmine ;  if  not,  with  BEALE'S 
carmine. 

539.  BELA  HALLER'S  Mixture  (MorphoJ.  Jahrb.,  xi,  p    321). 
— One  part  glacial  acetic  acid,  1  part  glycerin,  2  parts  water. 
Specially  recommended  for   the   central  nervous   system  of 
Mollusca  (Ehipidoglossa) .      A  maceration  of  thirty  to  forty 
minutes  may  be  sufficient,  the  cells  showing  less  shrinkage 
than  with  other  liquids. 

540.  Nitric  Acid. — Most  useful  for  the  maceration  of  muscle. 
The  strength  used  is  20  per  cent.      After  twenty-four  hours' 
maceration  in  this,  isolated  muscle-fibres  may  generally  be 
obtained  by  shaking  the  tissue  with  water   in   a  test-tube. 
Preparations  may  afterwards  be  washed  with  water  and  put 
up  in  strong  solution  of   alum,  in  which  they  may  be  pre- 
served for  a  long  time  (HOPKINS,  Proc.  Amer.  Soc.  of  Micro- 
scopists,  1890,  p.  165;  Zeit.  f.  iviss.  Mik.,  ix,  1,  1892,  p.  86). 

Maceration  is  greatly  aided  by  heat,  and  at  a  temperature 
of  40°  to  50°  C.  may  be  sufficiently  complete  in  an  hour 
((TAGE). 

541.  Nitric  Acid  and  Chlorate  of  Potash  (KiJHNE,  Ueber  die 
peripherischen     Endorgane,     etc.,    1862;     RANVIKR,     Traite, 


318  CHAPTER   XXIV. 

p.  79). — Chlorate  of  potash  is  mixed,  in  a  watch-glass,  with 
four  times  its  volume  of  nitric  acid.  A  piece  of  muscle  is 
buried  in  the  mixture  for  half  an  hour,  and  then  agitated 
with  water  in  a  test-tube,  by  which  means  it  entirely  breaks 
up  into  isolated  fibres. 

542.  Nitric  and  Acetic  Acid  (APATHY,  Zeit.  f.  wiss.  Mik.,  x, 
1898,  p.  49). — 3  vols.  glacial  acetic  acid,  3  of  nitric  acid,  and 
20  each  of  water,  glycerin,  and  absolute  alcohol.      Macerate 
leeches  for  twenty-four  hours,  and  bring  them  into  70  per 
cent,  alcohol,  in  which  they  swell ;    then  after  twenty-four 
hours,  50  per   cent,  glycerin,   changed   till    the   acid   is   re- 
moved. 

543.  Sulphuric  Acid  (RANVIER,  Traite,  p.  78). — Macerate  for 
twenty-four  hours  in  30  grms.  of  water,  to  which  are  added 
4  to  5  drops  of  concentrated  sulphuric  acid.      Agitate.      For 
nasal  mucosa,  crystalline,  retina,  etc. 

ODENIUS  found  very  dilute  sulphuric  acid  to  be  the  best 
reagent  for  the  study  of  nerve  endings  in  tactile  hairs.  He 
macerated  hair-follicles  for  from  eight  to  fourteen  days  in  a 
solution  of  from  3  to  4  grains  of  "  English  sulphuric  acid  " 
to  the  ounce  of  water. 

Hot  concentrated  sulphuric  acid  serves  to  dissociate  horny 
epidermic  structures  (horn,  hair,  nails). 

544.  Oxalic  Acid. — Maceration  for  many  days  in  concen- 
trated solution  of  oxalic  acid  has  been  found  useful  in  the 
study  of  nerve  endings. 

545.  SCHIEFFERDECKER'S   Methyl    Mixture  (for   the   retina) 
(Arch.  f.  mik.  Anat.,  xxviii,  1886,  p.  305). — Ten  parts  of  gly- 
cerin, 1   part  of   methyl   alcohol,  and   20  parts  of  distilled 
water.      Macerate  for  several  days  (perfectly  fresh  tissue). 

546.  Lysol  (REINKE,  Anat.  Anz.,  viii,  1892,  p.  582  ;  Zeit.  f. 
wiss.  Mik.,  x,  2,  1893,  p.  224). — Lysol  is  an  industrial  pro- 
duct consisting  in  a  solution   of    cresol    in  a  neutral  soap. 
REINKE  uses  a  10  per  cent,  solution  in  distilled  water  or  in 
water  containing  alcohol  and  glycerin.      Spermatozoa  of  the 
rat  or  cortical  cells  of  hairs  are  said  to  be  resolved  into  fibrils 


MACERATION  AND    DIGESTION.  319 

in  a  few  minutes.      Epithelial  cells  of  salamandra  are  said  to 
be  dissociated  instantaneously. 


Digestion. 

547.  BEALE'S  Digestion  Fluid  (Archives  of  Medicine,  i,  1858, 
pp.  296 — 316). — The  mucus  expressed  from  the  stomach 
glands  of  the  pig  is  rapidly  dried  on  glass  plates,  powdered, 
and  kept  in  stoppered  bottles.  It  retains  its  properties  for 
years.  Eight  tenths  of  a  grain  will  dissolve  100  grains  of 
coagulated  white  of  egg. 

To  prepare  the  digestion  fluid,  the  powder  is  dissolved  in 
distilled  water,  and  the  solution  filtered.  Or  the  powder  may 
be  dissolved  in  glycerin.  The  tissues  to  be  digested  may  be 
kept  for  some  hours  in  the  liquid  at  a  temperature  of  100°  F. 
(37°  C.). 

548.  BRUCKE'S  Digestion  Fluid  (from  CARNOY'S  Biologie  cellu- 
laire,  p.  94). 

Glycerinated  extract  of  pig's  stomach        .      1  vol. 
0*2  per  cent,  solution  of  HC1  .          .          .3  vols. 
Thymol,  a  few  crystals. 

549.  BICKFALVI'S  Digestion  Fluid  (Centrabl.f.  d.  med.  Wiss., 
1883,  p.  833). — One  grm.  of  dried  stomachal  mucosa  is  mixed 
with  20  c.c.  of  0'5  per  cent,  hydrochloric  acid,  and  put  into 
an  incubator  for  three  or  four  hours,  then  filtered.      Macerate 
the  tissue  in  the  solution  for  not  more  than  half  an  hour  to 
an  hour. 

550.  KUSKOW'S  Digestion  Fluid   (Arch.  f.  mik.  Anat.,  xxx, 
p.  32;  Zeit.f.  wiss.  Mik.,  iv,  3,  1887,  p.  384).— One  part  of 
pepsin  dissolved  in  200  parts  of  3  per  cent,  solution  of  oxalic 
acid.       The  solution    should   be   freshly  prepared,    and   the 
objects  (sections  of  hardened  Ligamentum  Nuchae)  remain  in 
it  at  the  ordinary  temperature  for  ten  to  forty  minutes. 

551.  SCHIEFFERDECKER'S  Pancreatin  Digestion  Fluid  (Zeit.f. 
wiss.  Mik.,  iii,  4,  1886,  p.  483). — A  saturated  solution  of  the 
"  Pankreatinum  siccum,"  prepared  by  Dr.  Witte,  Eostock,  is 


320  CHAPTER   XXIV. 

made  in  distilled  water,  cold,  and  filtered.  Pieces  of  tissue 
(epidermis)  are  macerated  in  it  for  three  to  four  hours  at 
.about  body  temperature. 

552.  KUHNE'S  Trypsin  Methods  (see  Unters.  a.  d.  Phys.  Inst.  Univ. 
Heidelberg,  i,  2,  1877,  p.  219).— Very  complicated. 

553.  GEDOELST'S  Methods  (see  La  Cellule,  iii,  1887,  p.  117,  and  v, 
1889,  p.  126;  also  Zeit.f.  wiss.  Mile.,  vii,  i,  1890,  p.  57). 


CHAPTER   XXV. 

CORROSION,    DECALCIFICATION,   DESILICIKICATION,   AND 
BLEACHING. 

Corrosion. 

554.  Caustic  Potash,  Caustic  Soda,  Nitric  Acid. — Boiling,  or 
long  soaking  in  a  strong  solution  of  either  of   these,  is  an 
efficient  means  of  removing  soft  parts  from  skeletal  structures 
(appendages  of  Arthropods,  spicula  of  sponges,  etc.). 

555.  Eau  de  Javelle  (Hypochlorite  of  Potash)  (NOLL'S  Method, 
Znol.Anzeig.,  122,  1882,  p.  528).— The  usual  method  of  pre- 
paring the  skeleton  of  siliceous  sponges  and  similar  structures 
by  corroding  away  the  soft  parts  by  means  of  caustic  potash 
has  many  disadvantages,  of  which  a  principal  one  is  that  the 
spicula  are  not  preserved  in  their  normal  positions.      NOLL 
proceeds  as  follows  : — A  piece  of  sponge  is  brought  on  to  a 
slide  and  treated  with  a  few  drops  of  eau  de  Javelle,  in  which 
it   remains   until   all   soft   parts  are  dissolved.      (With  thin 
pieces  this  happens  in  twenty  to  thirty  minutes.)      The  pre- 
paration is  then  cautiously  treated  with  acetic  acid,  which 
removes  all  precipitates  that  may  have  formed,  and  treated 
with  successive  alcohols  and  oil  of  cloves,  and  finally  mounted 
in  balsam. 

The  same  process  is  stated  to  be  applicable  to  calcareous 
structures.  I  feel  convinced,  however,  that  if  the  structures 
are  delicate,  they  will  suffer,  or  be  entirely  destroyed. 

556.  Eau  de  Labarraque  (Hypochlorite  of  Soda)  may  be  used 
in  the  same  way  as  eau  de  Javelle.      Looss  (Zool.  Anz.,  1885, 
p.  333)   finds  that  either  of  these   solutions  will  completely 
dissolve  chitin  in  a  short  time  with  the  aid  of  heat.      For  this 

21 


322  CHAPTER   XXV. 

purpose  the  commercial  solution  should  be  taken  concentrated 
and  boiling  (see  also  §  576). 

If  solutions  diluted  with  4  to  6  volumes  of  water  be  taken, 
and  chitinous  structures  be  macerated  in  them  for  twenty- 
four  hours  or  more,  according  to  size,  the  chitin  is  not  dis- 
solved, but  becomes  transparent,  soft,  and  permeable  to 
staining  fluids,  aqueous  as  well  as  alcoholic.  The  most 
delicate  structures,  such  as  nerve  endings,  are  stated  not  to 
be  injured  by  the  treatment.  The  method  is  applicable  to 
Nematodes  and  their  ova  (objects  well  known  for  the  re- 
sistance they  oppose  to  ordinary  reagents),  and  also  to  the 
removal  of  the  albumen  from  ova  of  Amphibia,  etc. 

557.  ALTMANN'S  Corrosion  Method  (Arch.f.  mik.  Anat.,  1879,  p.  471). 
— See  previous  editions,  or  Journ.  Roy.  Mic.  Soc.,  1879,  p.  610. 

On  injections  for  corrosions  see  EEJSEK,  Bibliogr.  Anat.,  iv,  1897,  p.  229. 

Decalcification. 

558.  Decalcification. — In  order  to  obtain  the  best  results,  it 
is  important  to  employ  only  material  that  has  been  duly  fixed 
and  hardened,  and  it  is  well  not  to  put  too  much  confidence 
in  reagents  that  are  said  to  have  the  property  of  hardening 
and  decalcifying  fresh  material  at  the  same  time  (Fisn,  Kef. 
Eandb.  Med.  Sci.,  Supp.,  p.  425). 

ROUSSEAU  (Zeit.  f.  wiss.  Mik.,  xiv,  1897,  p.  207)  imbeds 
fixed  material  in  celloidin,  brings  it  into  85  per  cent,  alcohol, 
decalcifies  in  a  very  acid  mixture  (15  to  40  percent,  of  nitric 
acid  in  alcohol),  washes  out  the  acid  in  alcohol  containing 
precipitated  carbonate  of  lime,  then  cuts  sections.  This  for 
Porifera,  corals,  Echinoderms,  etc.  Tissues  are  said  to  be 
well  preserved. 

559.  Decalcification  of  Bone. — I  take   the   following   from 
BUSCH,  Arch.  f.  mik.  Anat.,  xiv,  1877,  p.   481  ;  see  also  the 
paper  of  HAUG,  in  Zeit.  f.  wiss.  Mik.,  viii,  i,  1891,  p.  1. 

The  most  widely  used  agent  for  decalcification  is  hydro- 
chloric acid.  Its  action  is  rapid,  even  when  very  dilute,  but 
it  has  the  disadvantage  of  causing  serious  swelling  of  the 
tissues.  To  remedy  this,  chromic  acid  may  be  combined  with 
it,  or  alcohol  may  be  added  to  it.  Or  a  3  per  cent,  solution 


CORROSION,  DECALCIFICAT10N,  ETC.  323 

of  the  acid  may  be  taken  and  have  dissolved  in  it  10  to  15 
per  cent,  of  common  salt.  Or  (WALDEYER)  to  a  TTJVo  Per 
cent,  solution  of  chloride  of  palladium  may  be  added  -j^  of 
its  volume  of  HC1. 

Chromic  acid  is  also  much  used,  but  has  a  very  weak  de- 
calcifying action  and  a  strong  shrinking  action  on  tissues. 
For  this  latter  reason  it  should  never  be  used  in  solutions  of 
more  than  1  per  cent,  strength,  and  for  delicate  structures 
much  lower  strengths  must  be  taken. 

Phosphoric  acid  has  been  recommended  for  young  bones. 

Acetic,  lactic,  and  pyroligneous  acids  have  considerable 
decalcifying  power,  but  cause  great  swelling.  Picric  acid 
has  a  very  slow  action,  and  is  only  suitable  for  very  small 
structures. 

560.  Nitric  Acid  (Buscn,  loc.  cit.). — To  all  other  agents 
BUSCH  prefers  nitric  acid,  which  causes  no  swelling  and  acts 
most  efficaciously,  whilst  at  the  same  time  it  does  not 
injuriously  attack  tissue-elements. 

One  volume  of  chemically  pure  nitric  acid  of  sp.  gr.  1*25 
is  diluted  with  10  vols.  water.  It  may  be  used  of  this 
strength  for  very  large  and  tough  bones  ;  for  young  bones  it 
may  be  diluted  down  to  1  per  cent. 

Fresh  bones  are  first  laid  for  three  days  in  95  per  cent, 
alcohol ;  they  are  then  placed  in  the  nitric  acid,  which  is 
changed  daily,  for  eight  or  ten  days.  They  must  be  removed 
as  soon  as  the  decalcification  is  complete,  or  else  they  will 
become  stained  yellow.  When  removed  they  are  washed 
for  one  or  two  hours  in  running  water  and  placed  in  95  per 
cent,  alcohol.  This  is  changed  after  a  few  days  for  fresh 
alcohol. 

Young  and  foetal  bones  may  be  placed  in  the  first  instance 
in  a  mixture  containing  1  per  cent,  bichromate  of  potash  and 
tk  per  cent,  chromic  acid,  and  decalcified  with  nitric  acid  of 
1  to  2  per  cent.,  to  which  may  be  added  a  small  quantity  of 
chromic  acid  (^  per  cent.)  or  bichromate  of  potash  (1  per 
cent.).  By  putting  them  afterwards  into  alcohol  the  well- 
known  green  stain  is  obtained. 

561.  Nitric  Acid  and  Alcohol. — 3  per  cent,  of  nitric  acid  in 
70  per  cent,  alcohol.  Soak  specimens  for  several  days  or 


324  CHAPTER   XXV. 

weeks.  I  do  not  know  who  first  recommended  this  admir- 
able medium  (MAYER  has  long  used  5  per  cent,  acid  in  90  per 
cent,  alcohol).  Pure  nitric  acid,,  even  if  weak,  readily  exer- 
cises a  gelatinising  action  on  bone ;  whilst  the  addition  of 
alcohol  (or  of  alum)  counteracts  this  action  (FisH,  Ref. 
Handb.  Med.  Sci.,  Supp.,  p.  425). 

THOMA  (Zeit.  f.  wiss.  Mik.,  viii,  2,  1891,  p.  191)  takes  five 
vols.  of  95  per  cent,  alcohol  and  1  volume  pure  concentrated 
nitric  acid.  Leave  bones  in  this  mixture,  changing  the  liquid 
every  two  or  three  days,  until  thoroughly  decalcified,  which 
should  happen,  even  with  large  bones,  in  two  or  three  weeks. 
Wash  out  until  every  trace  of  acid  is  removed  (i.  e.  for  some 
days  after  no  acid  reaction  is  obtained  with  litmus  paper)  in 
95  per  cent,  alcohol  containing  an  excess  of  precipitated 
chalk.  This  may  take  eight  to  fourteen  days,  after  which 
the  tissues  will  stain  well  and  may  be  treated  as  desired. 

562.  Nitric  Acid  and  Alum  (G-AGE,  quoted  from  FISH,  Zoc, 
cit.  last  §). — A  saturated  aqueous  solution  of  alum  is  diluted 
with  an  equal  volume  of  water,  and  to  each  100  c.c.  of  the 
dilute  solution  is  added  5  c.c.  of  strong  nitric  acid.  Change 
every  two  or  three  days,  until  the  decalcification  is  complete. 
For  teeth  this  is  said  to  be,  perhaps,  a  better  decalcifier  than 
the  alcohol  mixture. 


563.  Hydrochloric  Acid  (see  above,  §  559). — RANVIEE  says  that  it 
may  be  taken  of  50  per  cent,  strength,  and  then  has  a  very  rapid  action. 
To  counteract  the  swelling  action  of  the  acid,  sodium  chloride  may  be  added 
(VON  EBNER),  see  HAUG'S  paper  quoted  §  559.     He  takes  either  100  c.c. 
cold  saturated  solution  of  sodium  chloride  in  water,  100  c.c.  water,  and 
4  c.c.  hydrochloric  acid.     Preparations  to  be  placed  in  this,  and  1  to  2  c.c. 
hydrochloric  acid  added  daily  until  they  are  soft.     Or,  2*5  parts  of  hydro- 
chloric acid,  500  of  alcohol,  100  of  water,  and  2*5  of  sodium  chloride. 
HAUG  prefers  the  proportions  of  TO  to  5'0  of  acid,  70  of  alcohol,  30  of 
water,  and  0'5  of  salt. 

564.  Hydrochloric  Acid  and  Chromic  Acid  (BAYEKL,  Arch.  f.  mik. 
Anat.,  1885,  p.  35). — Equal  parts  of  3  per  cent,  chromic  acid  and  1  per  cent, 
hydrochloric  acid.     For  ossifying  cartilage.     HAUG  recommends  equal  parts 
of  1  per  cent,  hydrochloric  acid  and  1  per  cent,  chromic  acid  (loc.  cit.). 

565.  Hydrochloric  Acid  and  Glycerin.— Glycerin,  95 ;  hydrochloric 
acid,  5  (S^uiRE's  Methods  and  Formulae,  p.  12). 


CORROSION,    DECALCIFIOATION,   ETC.  325 

566.  Picric  Acid  should  be  taken  saturated,  and  changed  frequently. 
Its  action  is  weak. 

Picro-sulphuric  acid  should  of  course  be  avoided  on  account  of  the 
formation  of  gypsum. 

Picro-nitric  or  Picro-hydrochloric  Acid. — MAYER  points  out  that  their 
action  is  very  rapid,  and  that  the  copiously  evolved  C02  often  produces, 
mechanically,  lesions  in  tissues;  so  that  in  many  cases  chromic  acid  is  to  be 
preferred,  the  more  so  as  it  more  effectually  hinders  any  collapsing  of  the 
structures  that  might  result  from  the  withdrawal  of  their  supporting  cal- 
careous elements. 

567.  Phosphoric  Acid.— 10  to  15  per  cent.  (HAUG,  loc.  cit.  in  §  559). 
Somewhat  slow,  staining  not  good. 

568.  Lactic  Acid. — 10  per  cent,  or  more.     Fairly  rapid,  preserves  well, 
and  may  be  recommended  (HAUG,  loc.  cit.). 

569.  Chromic  Acid  is  employed  in  strengths  of  from  O'l  per  cent,  to 
2  per  cent,  (but  see  §  559),  the  maceration  lasting  two  or  three  weeks  (in 
the  case  of  bone).     It  is  better  to  take  the  acid  weak  at  first,  and  increase 
the  strength  gradually.     In  any  way  the  action  is  extremely  slow,  and  it  is 
therefore  better  to  take  one  of  the  mixtures  of  chromic  acid  with  a  more 
energetic  agent. 

570.  Chromic  and  Nitric  Acid. — FOL  takes  70  volumes  of  1  per  cent, 
chromic  acid,  3  of  nitric  acid,  and  200  of  water  (Lehrb.,  p.  112). 

Even  with  the  addition  of  nitric  or  hydrochloric  acid  the  action  is  exces- 
sively slow,  frequently  requiring  months  to  be  complete. 

571.  Chromo-aceto-osmic  Acid  (VAN  VEB  STRICBT,  Arch.  Biol., 
ix,  1889,  p.  29;  and  SCHAFFEK,  Zeit.  f.  wiss.  Mik.,'x,  1893, 
p.  179). — Objects  to  be  left  in  it  for  months,  the  liquid  being 
changed  at  first  every  two  days,  afterwards  less  frequently. 
Structure  well  preserved. 

572.  Arsenic  Acid. — 4  per  cent,  aqueous  solution,  used  at  a  tempera- 
ture of  30°  to  40°  C.  (SQUIBE'S  Methods  and  Formulae,  etc.,  p.  11). 

573.  Phloroglucin  with  Acids  (ANDEEK,  Centralbl.  f.  d.  med. 
Wiss..,  xii,  xxxiii,  pp.  193,  579  ;  Intern.  Monatsscnr.,  i,  p.  350  ; 
Zeit.  f.  wiss.  Mik.,  1885,  pp.  375,  539  ;  Journ.  Boy.  Mic. 
Soc.,  1887,  p.  504;  HACG,  Zeit.  f.  wiss.  Mile.,  viii,  1,  1891, 
p.  8;  FERBERI,  ibid.,  ix,  2,  1892,  p.  236;  Bull.  R.  Accad. 
Med.  di  Boma,  1892,  p.  67). — This  is  the  most  rapid  method 
of  any.  Phloroglucin  by  itself  is  not  a  solvent  of  lime  salts  ; 


326  CHAPTER    XXV. 

its  function  in  the  mixtures  given  below  is  so  to  protect  the 
organic  elements  of  tissues  against  the  action  of  the  mineral 
acids  that  these  can  be  used  in  a  much  more  concentrated 
form  than  would  be  otherwise  advisable. 

HAUG  advises  the  following  procedure  : — Bring  one  grm.  of 
phloroglucin  into  10  c.c.  of  pure,  not  fuming  nitric  acid  (1*4 
sp.  gr.),  and  warm  very  slowly  and  carefully  with  gentle 
agitation.  There  is  formed  a  clear  solution  of  (presumably) 
a  nitrate  of  phloroglucin.  Dilute  the  solution  with  100  c.c. 
of  distilled  water,  and  add  10  c.c.  of  nitric  acid.  This  gives 
a  solution  containing  20  per  cent,  of  acid,  which  is  the  proper 
proportion.  More  water  may  be  added  to  the  solution,  to 
make  it  up  to  300  c.c.,  if  nitric  acid  be  also  added  in  the 
proportion  given.  But  the  dilution  must  not  be  carried 
beyond  this  point,  in  order  that  the  preservative  action  of  the 
phloroglucin  be  not  overmuch  weakened.  The  process  of 
decalcification  in  this  solution  is  extremely  rapid,  and  there- 
fore should  be  carefully  watched.  Foetal  and  young  bones 
become  quite  soft  in  half  an  hour ;  small  pieces  of  old  and 
hard  bones  (femur,  temporal  bone)  in  a  few  hours.  Teeth 
take  longer,  and  may  require,  if  time  be  an  object,  a  solution 
made  with  35  to  45  per  cent,  of  nitric  acid.  Wash  out  for  two 
days  in  running  water.  The  tissues  stain  well. 

The  solution  may  be  made  with  hydrochloric  acid  instead 
of  nitric  acid,  ;iO  per  cent,  of  acid  being  taken,  and  0*5  per 
cent  of  sodium  chloride  added. 

For  slow  decalcification  a  2  to  5  per  cent,  nitric  acid  solu- 
tion may  be  used,  or  a  mixture  containing  of  phloroglucin  1 
part,  nitric  acid  5,  alcohol  70,  and  distilled  water  30  parts. 

For  the  labyrinth,  FERRERI  advises  a  mixture  containing  1 
grm.  of  phloroglucin,  dissolved  with  the  aid  of  heat  in  10 
grms.  of  hydrochloric  acid  with  100  of  water,  200  of  70  per 
cent,  alcohol  being  added  after  cooling.  The  mixture  should 
be  changed  once  a  week  during  thirty  to  forty  days. 


Desilicifica  tion . 

574.  Hydrofluoric  Acid  (MAYEK,  Zool.  Anz.,  1881,  p.  593).— 
The  objects  are  brought  in  alcohol  into  a  glass  vessel  coated 
internally  with  paraffin  (otherwise  the  glass  would  be  corroded 


CORROSION,    DECALG1FICATION,    ETC.  327 

by  the  acid).  Hydrofluoric  acid  is  then  added  drop  by  drop 
(the  operator  taking  great  care  to  avoid  the  fumes,  which 
attack  mucous  membranes  with  great  energy) .  Small  pieces 
of  siliceous  sponges  will  be  completely  desilicified  in  a  few 
hours,  or  at  most  a  day.  The  tissues  do  not  suffer. 

This  dangerous  method  is  best  avoided  as  far  as  possible.  As  regards 
sponges,  I  would  point  out  that  if  well  imbedded  good  sections  may  be 
made  from  them  without  previous  removal  of  the  spicula.  The  spicula 
appear  to  be  cut;  probably  they  break  very  sharply  when  touched  by  the 
knife.  Kuives  are  of  course  not  improved  by  cutting  such  sections. 

ROUSSEAU  imbeds  the  objects  in  celloidin,  as  described  §  558,  then  brings 
the  block,  in  a  covered  caoutchouc  dish,  for  a  day  or  two  into  a  mixture  of 
50  c.c.  alcohol  and  20  to  30  drops  of  hydrofluoric  acid,  and  washes  out  the 
acid  with  alcohol  containing  carbonate  of  lithia  in  powder. 


Bleaching. 

575.  MAYKR'S  Chlorine  Method  (Hitth.  Zool.  Stat.  Neapel, 
ii,  1881,  p.  8). — Put  into  a  glass  tube  a  few  crystals  of 
qhlorate  of  potash,  add  two  or  three  drops  of  hydrochloric 
acid,  and  as  soon  as  the  green  colour  of  the  evolving  chlorine 
has  begun  to  show  itself,  add  a  few  cubic  centimetres  of 
alcohol  of  50  to  70  per  cent.  Now  put  the  objects  (which 
must  have  previously  been  soaked  in  alcohol  of  70  to  90  per 
cent.)  into  the  tube.  They  float  at  first,  but  eventually 
sink.  They  will  be  found  bleached  in  from  a  quarter  of  an 
hour  to  one  or  two  days,  without  the  tissues  having  suffered. 
Only  in  obstinate  cases  should  the  liquid  be  warmed  or  more 
acid  taken.  Sections  on  slides  may  be  bleached  in  this  way. 
Instead  of  hydrochloric  acid  nitric  acid  may  be  taken,  in 
which  case  the  active  agent  evolved  is  oxygen  instead  of 
chlorine. 

This  method  serves  both  for  removing  natural  pigments, 
such  as  those  of  the  skin  or  of  the  eyes  of  Arthropods,  and 
also  for  bleaching  material  that  has  been  blackened  by 
osmic  acid,  and,  according  to  renewed  experiments  of 
MAYER'S,  is  to  be  preferred  to  the  peroxide  of  hydrogen 
method. 

For  bleaching  chitin  of  insects,  not  alcohol  but  water 
should  be  added  to  the  chlorate  and  acid  (MAYER,  Arch.  Anat. 
Phys.,  1874,  p.  321). 


328  CHAPTER    XXV. 

576.  Eau  de  Labarraque.  Eau  de  Javelle  (see  §§  555,  556). — These 
are  bleaching  agents.  For  the  manner  of  preparing  a  similar  solution  see 
previous  editions,  or  Journ.  de  Microgr.,  1887,  p.  154,  or  Journ.  Roy.  Mic. 
Soc.,  1887,  p.  518.  Of  course  the  method  cannot  be  used  for  bleaching  soft 
parts  which  it  is  desired  to  preserve. 

577.  Peroxide  of  Hydrogen  (Oxygenated  Water)  (POUCHEI'S 
method,  M.  DUVAL,  Precis,  etc.,  p.  234). — Macerate  in 
glycerin,  to  which  has  been  added  a  little  oxygenated  water 
(5  to  6  drops  to  a  watch-glass  of  glycerin).  SOLGKR 
(Gentralbl.  mecL  Wiss.,  xxi,  1883,  p.  177)  takes  a  3  per  cent, 
solution  of  peroxide.  FURST  (Morph.  Arb.,  Schivalbe,  vi, 

1896,  p.  529)  points  out  that  after  a  time  it  macerates. 

The  method  serves  both  for  removing  pigments  and  for 
bleaching  osmic  and  chromic  material. 

578  Peroxide  of  Sodium  (CARAZZI,  Zool.  Anz.,  444,  1894, 
p.  135). — See  previous  editions. 

579.  Sulphurous  Acid. — Prof.  G-ILSON  writes  me  that  he 
finds  alcoholic  solution  of  sulphurous  anhydride  (S02)  very 
convenient  for  the  rapid  decoloration  of  bichromate  objects. 
A  few  drops  suffice.  MONCKEBERG  and  BETBE  ( Arch.  f.  m%k. 
Anat.,  liv,  1899,  p.  135;  Zeit.  f.  wins.  Mik.,  xvi,  2,  1899, 
p.  244)  obtain  the  acid  by  adding  to  10  c.c.  of  a  2  per  cent, 
solution  of  bisulphide  of  sodium  2  to  4  drops  of  concentrated 
hydrochloric  acid.  Objects  a.re  put  into  the  freshly  pre- 
pared solution  for  six  to  twelve  hours. 

580.  Permanganate  of  Potash.— ALFIEEI  (Monitore  Zool.  Hal,  viii, 

1897,  p.  57)  bleaches  celloidin   sections  of  the  choroid,  etc.,  for  eight  to 
twenty-four  hours  in  a  1 : 2000  solution  of  permanganate  of  potash,  then 
washes  them  out  for  a  few  hours  in  a  solution  of  oxalic  acid  of  1 :  300  strength, 
or  weaker. 


581.  GBENACHEE'S  Mixture  for  Eyes   of  Arthropods  and  other 
Animals  (AbJi.  nat.  Ges.  Halle-a.-S.,  xvi ;  Zeit.  f.  wiss.  Mik.,  1885,  p.  244). 
Glycerin  .  .  .  .  .1  part. 

80  per  cent,  alcohol         .  .  .  .2  parts. 

Mix  and  add  2  to  3  per  cent,  of  hydrochloric  acid. 

Pigments  [i.  e.  those  in  question]  dissolve  in  this  fluid,  and  so  doing  form 
a  stain  which  suffices  in  twelve  to  twenty-four  hours  for  staining  the  nuclei 
of  the  preparation. 


CORROSION,    DECALCIFICATION,   ETC.  329 

582.  Nitric  Acid.— PAEKEE  (Bull.  Mus.  Comp.  Zool,  Cambridge, 
U.S.A.,  1889,  p.  173 ;  see  Zeit.  f.  wits.  Mik.,  viii,  1,  1891,  p.  82)  says  that 
for  eyes  of  scorpions  the  usual  5  to  10  per  cent,  solutions  are  not  strong 
enough.  He  treats  sections,  fixed  to  the  slide  with  SCHALLIBAUM'S  medium, 
for  about  a  minute  with  a  solution  of  up  to  50  per  cent,  of  nitric  acid  in 
alcohol,  or,  still  better,  with  a  35  per  cent,  solution  of  a  mixture  of  equal 
parts  of  nitric  and  hydrochloric  acid  in  alcohol.  To  make  the  solution,  the 
acid  should  be  poured  slowly  into  the  alcohol  (not  vice  versa),  and  the 
mixture  kept  cool. 

JANDEE  (Zeit.  f.  wiss.  Mik.,  xv,  2,  1898,  p.  163)  takes  for  removal  of 
pigments  FOL'S  chromo-nitric  acid  (§  570) ;  twelve  to  forty-eight  hours  is 
enough  for  small  objects. 

584.  Caustic  Soda. — RAWITZ  (Leitfaden,  p.  29)  dissolves  the  pigment 
of  the  mantle  of  Lamellibranchiata  by  means  of  3  to  9  drops  of  officinal 
caustic  soda  solution  added  to  15  to  20  c.c.  of  96  per  cent,  alcohol. 


CHAPTER  XXVI. 

EMBBYOLOGICAL    METHODS. 

585.  Artificial  Fecundation. — This  practice,  which,   affords 
the  readiest  means  of  obtaining  the  early  stages  of  develop- 
ment of  many  animals,  may  be  very  easily  carried  out  in  the 
case  of  the  Amphibia  anura,  Teleostea,  Cyclostomata,  Echino- 
dermata,  and  many  Vermes  and  Coelenterata. 

In  the  case  of  the  Amphibia,  both  the  female  and  the 
male  should  be  laid  open,  and  the  ova  should  be  extracted 
from  the  uterus  and  placed  in  a  watch-glass  or  dissecting- 
dish,  and  treated  with  water  in  which  the  testes,  or,  better, 
the  vasa  deferentia,  of  the  male  have  been  teased. 

Females  of  Teleostea  are  easily  spawned  by  manipulating 
the  belly  with  a  gentle  pressure ;  and  the  milt  may  be 
obtained  from  the  males  in  the  same  way.  (It  may  occa- 
sionally be  necessary,  as  in  the  case  of  the  Stickleback,  to 
kill  the  male,  and  dissect  out  the  testes  and  tease  them.) 
The  spermatozoa  of  fish,  especially  those  of  the  Salmonidse, 
lose  their  vitality  very  rapidly  in  water;  it  is  therefore 
advisable  to  add  the  milt  immediately  to  the  spawned  ova, 
then  add  a  little  water,  and  after  a  few  minutes  put  the 
whole  into  a  suitable  hatching  apparatus  with  running 
water. 

Artificial  fecundation  of  Invertebrates  is  easily  performed 
in  a  similar  way.  It  is  sometimes  possible  to  perform  the 
operation  under  the  microscope,  and  so  observe  the  pene- 
tration of  the  spermatozoon  and  some  of  the  subsequent 
phenomena,  as  has  been  done  by  FOL,  the  HEKTWIGS, 
SELENKA,  and  others  for  the  Echinodermata  and  other  forms. 

586.  Superficial  Examination. — The  development   of  some 
animals,  particularly  some  Invertebrates,  may  be  to  a  certain 


EMBRYOLOGICAL    METHODS.  331 

extent  followed  by  observations  of  the  living  ova  under  the 
microscope.  This  may  usefully  be  done  in  the  case  of 
various  Teleosteans,  such  as  the  Stickleback,  the  Perch, 
Macropodus,  and  several  pelagic  forms,  and  with  Chironormis , 
Asellus  aquation*,  Ascidians,  Planorbis,  many  Ccelenterata, 
etc. 

Some  ova  of  Insecta  and  Arachnida  which  are  completely 
opaque  under  normal  conditions  become  transparent  if  they 
are  placed  in  a  drop  of  oil ;  if  care  be  taken  to  let  their 
surface  be  simply  impregnated  with  the  oil,  the  normal  course 
of  development  is  not  interfered  with  (BALBIANI). 

587.  Preparation  of  Sections. — Osmic  acid,  employed  either 
alone  or  in  combination  with  other  reagents,  is  an  excellent 
fixing  agent  for  small  embryos,  but  not  at  all  a  good  one  for 
large  ones.  It  causes  cellular  elements  to  shrink  somewhat, 
and  therefore  bring  out  very  clearly  the  slits  that  separate 
germinal  layers,  and  any  channels  or  other  cavities  that  may 
be  in  course  of  formation. 

In  virtue  of  its  property  of  blackening  fatty  matters, 
myelin  amongst  them,  it  is  of  service  in  the  study  of  the 
development  of  the  nervous  system. 

Chromic  acid  is  indispensable  for  the  study  of  the  external 
forms  of  embryos  ;  it  brings  out  elevations  and  depressions 
clearly,  and  preserves  admirably  the  mutual  relations  of  the 
parts ;  but  it  does  not  always  preserve  the  forms  of  cells 
faithfully,  and  is  a  hindrance  to  staining  in  the  mass. 

Picr?c  liquids  have  an  action  which  is  the  opposite  of  that 
of  osmic  acid  ;  they  cause  cellular  elements  to  swell  some- 
what, and  thus  have  a  tendency  to  obliterate  spaces  that 
may  exist  in  the  tissues.  But  notwithstanding  this  defect, 
the  picric  compounds,  and  especially  Kleinenberg's  picro- 
sulphuric  acid,  are  amongst  the  best  of  embryological  fixing 
agents. 

RABI.  (Zeit.  /.  wi«s.  Mik.,  xi,  2,  1894,  p.  165)  highly  re- 
commends for  embryos  of  Vertebrates,  and  also  for  other 
objects,  the  following  platino-subliinaifciipuxture  : 

Platinic  chloride,  1  per  cent,  solution    .     1  vol. 
Saturated  aqueous  sublimate  solution    .     1     ,, 
Distilled  water .  .  .  .  .2  vols. 

This  serves  for  a  large  number  of  blastoderms  and  young 


332  CHAPTER    XXVI. 

embryos  (Pisces,  Amphibia,  Aves,  Mammalia).  Advanced 
embryos  of  Teleostea  ought  to  be  fixed  in  the  warmed  mix- 
ture, in  order  to  avoid  rupture  of  the  muscles  and  shrinkage 
.of  the  chorda. 

RAUL'S  picro-sublimate  mixture  has  been  given  §61.  It 
is  recommended  especially  for  somewhat  advanced  embryos, 
such  as  embryo  chicks  from  the  third  or  fourth  day,  and 
other  embryos  of  a  similar  size. 

BOVERI  (Verh.  Physik.  Med.  G-es.  Wiirzburg,  xxix,  1895, 
p.  4),  in  order  to  imbed  and  cut  together  numbers  of  ova  of 
Echinoderms,  wraps  them  in  pieces  of  sloughed  epidermis  of 
Cryptobranchus  (of  course  other  Urodela  will  do).  SOBOTTA 
(Arch.  f.  mik.  Anat.,  1,  1897,  p.  31)  takes  pieces  of  amnios 
of  Mammalia. 

588.  Reconstruction  of  Embryos  from  Sections. — The  study  of 
a  series  of  sections  of  any  highly  differentiated  organism  is 
so  complicated  that  it  is  often  necessary  to  have  recourse  to 
elaborate  methods  of  geometrical  or  of  plastic  reconstruction 
in  order  to  obtain  an  idea  or  a  model  of  the  whole.  These 
methods  ha,ve  now  been  brought  to  so  high  a  degree  of  com- 
plexity that  a  volume  rather  than  a  paragraph  would  be 
necessary  to  describe  them.  See  BOKN,  "  Die  Plattenmodel- 
lirmethode,"  in  Arch.  f.  mik.  Anat.,  1883,  p.  591,  and  Zeit. 
f.  wiss.  Mik.,  v,  4,  1888,  p.  433;  STKASSKR,  in  Zeit.  f.  wiss. 
Mik.,  iii,  L>,  1886,  p.  179,  and  iv,  2  and  3,  pp.  168  and  830; 
KASTSCHENKO,  in  Zeit.  f.  wis*.  Mik.,  iv,  2  and  3,  1887,  pp. 
235-f>  and  353,  and  v,  2,  1888,  p.  173;  SCHAPEK  (ibid., 
xiii,  1897,  p.  446;  ALEXANDER,  ibid.,  p.  334,  and  xv,  1899, 
p.  446;  BORN  and  PKTER,  ibid.,  xv,  1,  p.  31  ;  JOHNSTON, 
Anat.  Anz.,  xvi,  1899,  p.  261.  For  a  method  of  FOL  (Lehrb., 
p.  35)  see  previous  editions. 

In  simple  cases  it  may  be  sufficient  to  adopt  the  plan 
described  by  SCHAFFER  (Zeit.f.  wiss.  Mik.,  vii,  3,  1890,  p.  342). 
Careful  outlines  of  the  sections  to  be  reconstructed  are  drawn 
on  tracing-paper  with  the  aid  of  the  camera  lucida,  super- 
posed, and  held  up  against  the  light  for  examination  by 
transparence.  YOSMAER  (Anat.  Anz.,  xvi,  1899,  p.  269) 
draws  on  plates  of  celluloid,  and  sets  them  up  in  a  rack  for 
examination. 

A    method   for    simple    graphic    reconstructions    without 


EMBRYOLOGICAL  METHODS.  333 

camera  drawings  is  described  by  WOOD  WORTH  (Zeit.  f.  wiss. 
Mik.,  xiv,  1,  1897,  p.  15)  :  1.  Draw  an  axial  line  of  the 
length  of  the  object  multiplied  by  the  magnification  em- 
ployed. 2.  Measure  with  a  micrometer  the  greatest  dia- 
meter of  each  section.  3.  Plot  these  diameters  down 
transversely  on  the  axial  line  at  distances  corresponding  to 
the  thickness  of  the  sections  multiplied  by  the  magnification. 
4.  Join  the  extremities  of  these  diameters ;  this  will  give  you 
an  outline  of  the  object.  5.  Measure  off  on  each  section  the 
nearest  and  farthest  limits  (from  the  margin)  of  the  organs 
to  be  filled  in,  and  plot  them  down  on  the  transverse  lines 
(3),  and  join  the  points  as  before,  i.  e.  from  section  to 
section  ;  this  will  give  you  the  outlines  of  the  organs. 

This  process  is  best  applicable  to  reconstruction  from  trans- 
verse sections,  but  it  can  be  applied  to  reconstruction  from 
sections  in  any  plane  if  the  object  can  be  provided  with  a 
plane  of  definition  at  right  angles  to  the  plane  of  section. 
This  may  be  established  by  cutting  off  one  end  of  the 
object,  or  the  like  (see  also  Orientation,  §§  133,  155). 


Mammalia. 

589.  Rabbit — Dissection, — For  the  study  of  the  early  stages 
the  ova  must  be  sought  for  in  the  tubse  a  certain  number  of 
hours  after  copulation.  The  dehiscence  of  the  follicles  takes 
place  about  ten  hours  after  the  first  coitus.  The  tubse  and 
cornua  having  been  dissected  out  should  be  allowed  to  cool, 
and  remain  until  the  muscular  contractions  have  ceased. 
Then,  with  the  aid  of  fine  scissors  or  a  good  scalpel,  all  th& 
folds  of  the  genital  duct  are  carefully  freed  from  their 
peritoneal  investment. 

The  tubae  are  then  (if  the  ova  are  still  within  them,  which 
is  the  case  up  to  the  end  of  the  third  day  after  coition)  laid 
out  on  a  long  slip  of  glass,  and  slit  up  longitudinally  by  means 
of  a  pair  of  fine,  sharp  scissors.  By  means  of  needles  and 
forceps  the  tubal  mucosa  is  spread  out  so  as  to  smooth  out 
its  folds  as  much  as  possible,  and  is  carefully  looked  over 
with  a  strong  lens  or  with  a  lower  power  of  the  microscope. 
When  the  ova  are  found,  a  drop  of  some  "  indifferent  "  liquid 
is  dropped  on  each,  and  it  is  carefully  taken  up  with  the 


334  CHAPTER    XXVI. 

point  of  a  scalpel,  a  cataract  needle,  or  a  small  pipette.  They 
may  be  examined  in  the  peritoneal  fluid  of  the  mother  if  the 
animal  lias  been  killed,  or  in  its  aqueous  humour,  or  in 
amniotic  liquid,  or  in  blood-serum,  or  in  KRONECKER'S  or 
other  artificial  serum. 

If  you  have  not  been  able  to  find  the  ova  with  the  lens  or 
the  microscope,  scrape  off  the  epithelium  of  the  tubal  mucosa 
with  a  small  scalpel,  mix  it  with  a  little  indifferent  liquid, 
and  look  for  the  ova  under  the  microscope  by  transmitted 
light. 

Another  method,  employed  by  KOLLIKER,  consists  in  in- 
jecting solution  of  MULLER  or  weak  osmic  acid  into  the  oviduct 
by  means  of  a  small  syringe,  and  collecting  the  liquid  that 
runs  out  in  a  series  of  watch-glasses,  in  which  the  ova  can 
very  easily  be  found  by  the  microscope. 

The  same  doe  may  be  made  to  serve  for  two  observations,  at  some  hours' 
or  days'  interval.  A  longitudinal  incision  of  8  to  10  centimetres'  length  is 
made  on  the  median  or  a  lateral  line  of  the  abdomen ;  an  assistant  keeps 
the  intestines  in  their  place ;  a  ligature  is  placed  at  the  base  of  one  of  the 
uterine  cornua,  beneath  the  neck,  and  a  second  ligature  around  the  meso- 
metrium  and  mesovarium.  The  ovary,  the  tuba,  and  the  cornu  of  that  side 
are  then  detached  with  scissors.  The  abdomen  is  then  closed  by  means  of 
a  few  sutures  passing  through  the  muscle-layers  and  the  skin.  The  animals 
support  the  operation  perfectly  well,  and  the  development  of  the  ova  of  the 
opposite  side  is  not  in  the  least  interfered  with.  When  it  is  desired  to  study 
these  the  animal  may  be  killed,  or  may  be  subjected  to  a  second  laparotomy 
if  it  be  desired  to  preserve  it  for  ulterior  observations. 

During  the  fourth,  fifth,  and  sixth  days  after  copulation 
the  ova  of  the  rabbit  are  free  in  the  uterine  cornua  ;  they  are 
easily  visible  to  the  naked  eye,  and  may  be  extracted  by  the 
same  manipulations  as  those  of  the  tubes.  After  the  sixth 
day  they  are  at  rest  in  the  uterus,  but  have  not  yet  contracted 
adhesions  with  the  mucosa,  so  that  they  can  still  be  extracted 
whole.  At  this  stage  the  parts  of  the  cornua  where  the  ova 
.are  lodged  are  easily  distinguishable  by  their  peculiar  aspect, 
the  ova  forming  eminences  of  the  size  of  a  pea.  The  cornua 
should  be  cut  up  transversely  into  as  many  segments  as  there 
are  eminences,  care  being  taken  to  have  the  ova  in  the  centre 
of  the  segments.  You  then  fix  each  segment  by  means  of 
two  pins  on  the  bottom  of  a  dissecting  dish,  with  the  meso- 
metrial  surface  downwards  and  the  ovular  eminence  upwards. 


EMBRYOLOGICAL   METHODS.  335 

The  dissecting-dish  is  then  filled  up  with  serum  or  liquid  of 
MULLER,  or  0*1  per  cent,  solution  of  osmic  acid,  or  KLEINEN- 
BERG'S  picro-sulphuric  acid,  or  nitric  acid,  or  acetate  of 
uranium  solution.  With  a  small  scalpel  a  longitudinal  in- 
cision is  made  on  the  surface  of  the  ovular  eminence,  not 
passing  deeper  than  the  muscular  layer;  the  underlying 
uterine  mucosa  is  then  gently  dilacerated  with  two  pairs  of 
small  forceps,  and  the  ovum  set  free  in  the  liquid. 

From  the  moment  the  ova  have  become  adherent  to  the 
uterine  mucosa  they  can  no  longer  be  extracted  whole.  The 
embryo  being  always  situated  on  the  mesometrial  surface,  the 
ovular  eminence  is  opened  by  a  crucial  incision,  and  the  strip 
of  mucosa  to  which  the  embryo  remains  adherent  is  fixed 
with  pins  on  the  bottom  of  the  dish.  ED.  v.  BENEDEN  (see 
Arch,  de  Biol.,  v,  fasc.  iii,  1885,  p.  378)  has  been  able  by 
operating  in  this  way  in  serum  of  Kronecker,  and  keeping 
the  whole  at  blood  temperature,  to  observe  the  circulation 
of  the  embryo  for  hours  together.  (If  this  be  desired  to  be 
done,  the  crucial  incision  should  not  be  too  extended,  so  as 
to  leave  the  terminal  sinus  intact.) 

RETTERER  (C.  R.  Soc.  de  Biol.,  1887,  p.  99)  advises  that 
for  ova  of  the  seventh  day  the  segment  of  uterus  containing 
them  be  opened  on  the  mesometrial  surface,  for  at  that  date 
no  adhesion  has  yet  been  contracted  with  that  side.  By 
running  in  liquid  of  Kleinenberg  by  means  of  a  pipette 
between  tne  ovum  and  the  free  surface  of  the  uterus,  the 
ovum  may  be  got  away  in  the  shape  of  a  closed  vesicle. 

590.  RABBIT  ;  Microscopic  Preparations. — In  order  to  make 
permanent  preparations  of  the  different  stages  of  fecundation 
and  segmentation,  v.  BENEDEN  (Arch,  de  Biol.,  i,  1,  1880,  p. 
149)  recommends  the  following  process  : — The  living  ovum  is 
brought  into  a  drop  of  1  per  cent,  osmic  acid  on  a  slide,  and 
thence  into  solution  of  Miiller  (or  bichromate  of  ammonia  or 
solution  of  Kleinenberg).  After  an  hour  the  liquid  is 
changed,  and  the  whole  is  put  into  a  moist  chamber,  where 
it  remains  for  two  or  three  days.  It  is  then  treated  with 
glycerin  of  gradually  increasing  strength,  and  at  last  mounted 
in  pure  glycerin  acidified  with  formic  acid.  Ova  may  be 
stained  after  careful  washing. 

In  order  to  bring  put  the  outlines  of  blastoderm  cells  the 


336  CHAPTER    XXVI. 

living  ovum  may  be  brought  into  one  third  per  cent,  solution 
of  nitrate  of  silver.  After  remaining  there  for  half  a  minute 
to  two  minutes,  according  to  the  age  of  the  vesicle,  it  is 
brought  into  pure  water  and  exposed  to  the  light.  The  pre- 
parations thus  obtained  are  instructive,  but  blacken  rapidly, 
and  cannot  be  permanently  preserved. 

After  the  end  of  the  third  day  the  blastodermic  vesicle 
can  be  opened  with  fine  needles,  and  the  blastoderm  washed, 
stained,  and  mounted  in  glycerin  or  balsam  ;  v.  BENEDEN 
has  also  obtained  good  preparations  by  means  of  chloride  of 
gold. 

For  embryonic  areas  and  more  advanced  embryos  KOLLIKER 
recommends  putting  the  ovum  into  0*5  per  cent,  solution  of 
osmic  acid  until  it  has  taken  on  a  somewhat  dark  tint,  which 
happens  in  about  an  hour,  and  then  treating  it  with  successive 
alcohols  for  several  hours.  If  the  ovum  be  adherent  to  the 
uterine  mucosa  the  portion  of  the  membrane  to  which  it  is 
fixed  should  be  left,  stretched  out  with  pins,  in  O'l  per  cent, 
solution  of  osmic  acid  for  from  four  to  six  hours.  The  blasto- 
dermic vesicle  can  then  easily  be  removed  and  further 
treated  as  before.  For  sections  KOLLIKER  fixes  with  osmic 
acid.  v.  BENEDEN  treats  the  ova  for  twenty-four  hours  with 
1  per  cent,  solution  of  chromic  acid,  then  washes  well,  and 
brings  them  through  successive  alcohols.  Chromic  acid  has 
the  advantage  of  hardening  thoroughly  the  vesicle,  and 
maintaining  at  the  same  time  the  epiblast  cells  perfectly 
adherent  to  the  zona  pellucida.  v.  BENEDEN  also  recommends 
the  liquid  of  Kleinenberg.  HENNEGUY  writes  that  he  fre- 
quently employs  it  for  embryonic  areas  and  embryos  of 
various  ages,  always  with  excellent  results.  Fol's  modifica- 
tion of  the  liquid  of  Flemming,  and  Ranvier  and  Vignal's 
osmic  acid  and  alcohol  mixture  (§  39  A)  also  give  excellent 
results.  For  staining,  HENNEGDY  recommends  borax -carmine, 
or  Delafield's  haematoxylin  for  small  embryos ;  for  large  ones 
he  found  that  his  acetic  acid  alum-carmine  was  the  only 
reagent  that  would  give  a  good  stain  in  the  mass.  I  think 
carmalum  is  now  indicated. 

For  sections  imbed  in  paraffin. 

See  also  WEYSSE,  Proc.  Amer.  Acad.  Arts  and  Sci.,  1894, 
p.  285  (blastodermic  vesicle  of  Sus  scrofa)  ;  SOBOTTA,  Arch, 
f.  mik.  Anat.,  xlv,  1895,  p.  15  (fecundation  and  segmentation 


EMBRIOLOGICAL    METHODS.  337 

of  the  ovum  of  the  mouse ;  fixation  in  FLEMM ING'S  weak 
mixture,  sections  stained  with  BENDA'S  iron  hsematoxylin) , 
an&Anat.  Hefte,  1  Abth.,  viii,  1897,  p.  476  (Rabbit;  fixation 
with  liquid  of  Flemming  or  picro-sublimate  with  2  per  cent, 
acetic  acid)  ;  BONNET,  ibid.,  ix,  1897,  p.  426  (Dog ;  fixation 
in  sublimate). 


Aves. 

591.  Superficial  Examination. — Instructions  on  this  head 
are  given  in  FOSTEIC  and  BALFOUB'S  Elements  of  Embryology. 
What  follows  here  is  given  merely  as  being  of  more  recent 
publication. 

If  it  be  desired  to  observe  a  living  embryo  by  transmitted 
light,  the  egg  should  be  opened  under  salt  solution,  as  de- 
scribed below.  A  little  of  the  white  is  then  removed  through 
the  window,  the  egg  is  lifted  out  of  the  liquid,  and  a  ring  of 
gummed  paper  is  placed  on  the  yolk  so  as  to  surround  the 
embryonic  area.  As  soon  as  the  paper  adheres  to  the  vitel- 
line  membrane,  which  will  be  in  a  few  minutes,  a  circular 
incision  is  made  in  the  blastoderm  outside  the  paper  ring. 
The  egg  is  put  back  into  the  salt  solution,  and  the  paper 
ring  removed,  carrying  with  it  the  vitelline  membrane  and 
the  blastoderm,  which  may  then  be  brought  into  a  watch 
glass  or  on  to  a  slide  and  examined  under  the  microscope 

(DUVAL)  . 

592.  Gerlach's  Window  Method  (Nature,  1886,  p.  497  ;  Journ.  Roy. 
Mic.  Soc.,  1886,  p.  359). — Remove  with  scissors  the  shell  from  the  small 
end  of  the  egg ;  take  out  a  little  white  by  means  of  a  pipette ;  the  blasto- 
derm will  become  placed  underneath  the  window  thus  made,  and  the  white 
that  has  been  taken  out  may  be  replaced  on  it.  Paint  the  margins  of  the 
window  with  gum  mucilage,  and  build  up  on  the  gum  a  little  circular  wall 
of  cotton  wool ;  place  on  it  a  small  watch  glass  (or  circular  cover-glass),  and 
ring  it  with  gum.  When  the  gum  is  dry,  the  cover  is  further  fixed  in  its 
place  by  means  of  collodion  and  amber  varnish,  and  the  egg  is  put  back  in 
its  normal  position  in  the  incubator.  The  progress  of  the  development  may 
be  followed  up  to  the  fifth  day  through  the  window. 

A  description  of  further  developments  of  this  method,  with  figures  of 
special  apparatus,  will  be  found  in  Anat.  Anz.,  ii,  1887,  pp.  583,  609 ;  see 
also  Zeit.f.  wiss.  Mik.,  iv,  3,  1887,  p.  369. 

593.  Preparation.— During  the  first  twenty-four  hours  of 
incubation  it  is  extremely  difficult  to  separate  the  blastoderm 

22 


338  CHAPTER    XXVI. 

from  the  yolk,  and  they  should  be  fixed  and  hardened  together. 
In  later  stages,  when  the  embryo  is  conspicuous,  the  blasto- 
derm can  easily  be  separated  from  the  yolk,  which  is  very 
advantageous.  To  open  the  egg,  lay  it  on  its  side  and  break 
the  shell  at  the  broad  end  by  means  of  a  sharp  rap  ;  then 
carefully  remove  the  shell  bit  by  bit  by  breaking  it  away 
with  forceps,  working  away  from  the  broad  end  until  the 
blastoderm  is  exposed.  The  egg  should  be  opened  in  salt 
solution,  then  lifted  up  a  little,  so  as  to  have  the  blastoderm 
above  the  surface  of  the  liquid;  the  blastoderm  is  then  treated 
with  some  fixing  solution  dropped  on  it  from  a  pipette  (1  per 
cent,  solution  of  osmic  acid,  or  Ranvier  and  Vignal's  osmic 
acid  and  alcohol  mixture,  iodised  serum,  solution  of  Kleinen- 
berg,  10  per  cent,  nitric  acid,  etc.).  By  keeping  the  upper 
end  of  the  pipette  closed,  and  the  lower  end  in  contact  with 
the  liquid  on  the  blastoderm,  the  blastoderm  may  be  kept 
well  immersed  for  a  few  minutes,  and  should  then  be  found  to 
be  sufficiently  fixed  to  be  excised.  (Of  course,  if  you  prefer 
it,  you  can  open  the  egg  in  a  bath  of  any  fixing  liquid  [10  per 
cent,  nitric  acid  being  convenient  for  this  purpose]  of  such  a 
depth  as  to  cover  the  yolk ;  and  having  exposed  the  blasto- 
derm, leave  it  till  fixed  [fifteen  to  twenty  minutes]  ;  but  I 
think  the  procedure  above  described  will  generally  be  found 
more  convenient.) 

The  egg  is  put  back  into  the  salt  solution,  and  a  circular 
incision  made  round  the  embryonic  area.  The  blastoderm 
may  then  be  floated  out  and  got  into  a  watch  glass,  in  which 
it  may  be  examined,  or  may  be  brought  into  a  hardening 
liquid. 

Before  putting  it  into  the  hardening  fluid,  the  portion  of 
vitelline  membrane  that  covers  the  blastoderm  should  be 
removed  with  forceps  and  shaking. 

Fixation  in  10  per  cent,  nitric  acid  has  the  advantage  of 
greatly  facilitating  the  separation  of  the  blastoderm.  The 
acid  should  be  allowed  to  act  for  ten  minutes,  after  which  it 
is  well  to  bring  the  preparation  into  2  per  cent,  solution  of 
alum  (cf.  HOPMANN,  Zeit.  f.  wiss.  Mik.,  x,  4,  1893,  p.  485). 

In  order  to  counteract  the  turning  up  of  the  edges  of  the 
blastoderm  that  generally  happens  during  the  process  of 
hardening,  it  is  well  to  get  the  blastoderm  spread  out  on  the 


EMBRYOLOGICAL    METHODS.  339 

convex  surface  of  a  watch  glass,  and  leave  it  so  during  the 
hardening. 

For  hardening,  HENNEQUY  prefers  the  osmic  acid  and  alcohol 
mixture  of  Ranvier  and  Vignal,  or  Flemming's  mixture  fol- 
lowed by  successive  alcohols. 

Stain  and  imbed  by  the  usual  methods. 

Up  to  about  the  fiftieth  hour  embryos  may  be  mounted 
entire  in  glycerin  or  balsam. 

594.  M.  DUVAI/S  Orientation  Method  (Ann.  Sc.  Nat.,  1884, 
p.  3). — In  the  early  stages  of  the  development  of  the  ova  of 
Aves,  before  the  appearance  of    the   primitive  streak,  it  is 
difficult  to  obtain  a  correct  orientation  of  the  hardened  cica- 
tricula, so  as  to  be  able  to  make  sections  in  any  desired  direc- 
tion.     DUVAL,  starting  from  the  fact  that  during  incubation 
the  embryo  is  almost  always  found  to  be  lying  on  the  yolk  in 
such  a  position  that  the  big  end  of  the  egg  is  to  the  left,  and 
the  little  end  to  the  right  of  it,  marks  the  position  of  the 
blastoderm  in  the  following  way. 

With  a  strip  of  paper  5  millimetres  wide  and  50  millimetres 
long  you  construct  a  sort  of  triangular  bottomless  box.  You 
lay  this  on  the  yolk,  enclosing  the  cicatricula  in  such  a  posi- 
tion that  the  base  of  the  triangle  corresponds  to  what  will  be 
the  anterior  region  of  the  embryo,  and  its  apex  to  the  posterior 
region  ;  that  is  to  say,  if  the  big  end  of  the  egg  is  to  your 
left,  the  apex  of  the  triangle  will  point  towards  you.  You 
now,  by  means  of  a  pipette,  fill  the  paper  triangle  with  0*3 
per  cent,  solution  of  osmic  acid.  As  soon  as  the  preparation 
begins  to  darken  you  put  the  whole  egg  into  weak  chromic  acid, 
remove  the  white,  and  put  the  rest  into  clean  chromic  acid 
solution  for  several  days.  After  hardening  you  will  find  on 
the  surface  of  the  yolk  a  black  triangular  area,  which  encloses 
the  cicatricula  and  marks  its  position ;  you  cut  out  this  area 
with  scissors  and  a  scalpel,  and  complete  the  hardening  with 
chromic  acid  and  alcohol. 

See  also  the  method  of  HIROTA,  Journ.  Roy.  Mic.  Soc., 
1895,  p.  118. 

595.  KIONKA'S   Orientation  Method    (Anat.  Hefte,   I   Abth., 
iii,  1894,  p.  414;  Zeit.  f.  wis*.  Mik.,  xi,  2,  1894,  p.  250).- 
Open  the  egg  under  salt  solution,  free  it  from  the  shell  and 


340  CHAPTER   XXVI. 

albumen,  and  mark  the  poles  by  sticking  into  it,  at  about  a 
centimetre  from  the  blastoderm,  two  hedgehog  spines,  the 
one  at  the  obtuse  end  being  marked  with  a  red  thread. 
Put  the  whole  for  ten  minutes  into  water  at  90°  C.,  then 
bring  into  70  per  cent,  alcohol,  and  after  twenty-four  hours 
cut  out  the  blastoderm  and  a  little  yolk  round  it  in  the 
shape  of  an  isosceles  triangle  whose  base  marks  the  anterior 
end  of  the  blastoderm.  Paraffin  sections  stained  with  borax- 
carmine,  washed  out  with  acid  alcohol  containing  one  drop 
of  concentrated  solution  of  Orange  Gr  for  each  5  c.c.,  which 
stains  the  yolk. 

596.  VIALLETON'S  Method  (Anat.  Anzeig.,  vii,  1892,  pp.  624—627; 
Journ.  Roy.  Mic.  Soc.,  1892,  p.  889). — Egg  opened  in  salt  solution,  blasto- 
derm excised  and  removed  to  a  glass  plate,  then  treated  with  1  per  cent, 
nitrate  of  silver  solution,  washed  with  water,  and  put  into  70  per  cent, 
alcohol    for   six    to   twelve    hours   in   the   dark.     Borax-carmine,  alcohol, 
damar. 

597.  BOHM  and  OPPEL  (Taschenbuch,  1896,  p.  80)  fix  ova  with  fairly 
large  embryos  in  a  mixture  of  20  parts  3  to  5  per  cent,  nitric  acid,  and  1  to 
2  parts  1  per  cent,  silver  nitrate. 


Reptilia. 

598.  General  Directions. — The  methods  described  above  for 
birds   are   applicable   to  reptiles.      During  the  early  stages 
the   blastoderm  should   be   hardened   in  situ  on   the   yolk; 
later   the   embryo   can   be   isolated,   and  treated   separately 
with  fixatives,  etc. 

599.  Special  Cases. — MITSUKURI  (Journ.  Coll.  Sc.  Japan,  vi, 
1894,  p.  229)   fixes  embryos  of  tortoises  chiefly  with  picro- 
sulphuric  acid.      To  study  the   blastoderm   he   removes  the 
whole  of  the  shell  and  as  much  as  possible  of  the  albumen, 
marks   the   place   where   the   blastoderm   lies   with    a    hair, 
brings  the  whole,  with  the  blastoderm  uppermost,  into  the 
fixative,  and  after  a  few  hours  cuts  out  the  blastoderm  and 
further  hardens   it   by   itself.       Young    embryos    generally 
adhere  to  the  shell  and  can,  therefore,  be  fixed  in  a  piece  of 
it  made  to  serve  as  a  watch  glass,  then  after  half  an  hour 
can  be  removed  from  it  and  further  hardened  alone.      If  the 


EMBRYOLOGTCAL    METHODS.  341 

embryonal  membranes  have  been  formed,  the  shell  may  be 
scraped  away  at  some  spot  and  there  treated  with  picro- 
sulphuric  acid  until  a  small  hole  is  formed  ;  then  by  working 
away  from  this  spot,  by  means  of  scraping  and  dropping 
acid  on  to  it,  the  whole  of  the  shell  may  be  removed. 

WILL  (Zool.  Jahrb.,  Abth.  Morph.,  vi,  1892,  p.  8)  opens 
ova  of  Platydactylus  in  the  fixative  (chiefly  chromic  acid,  or 
chromo-aceto-osmic  acid  with  very  little  osmic  acid)  and 
hardens  the  embryos  on  the  yolk ;  so  also  for  Cistudo  and 
Lacerta  (1893  and  1895).  MEHNERT  (Anat.  Anz.,  xi,  1895, 
p.  257)  does  not  approve  of  these  methods ;  for  his  own  see 
Morph.  Arb.  Schwalbe,  i,  1891,  p.  370. 

STRAHL  (Arch.  Anat.  Phys.,  Anat.  Abth.,  1881,  p.  123) 
uses  picro-sulphuric  acid  for  Lacerta. 

KUPFFER  (op.  cit.,  1882,  p.  4)  removes  ova  of  Lacerta, 
Emys,  Coluber,  etc.,  from  the  oviduct,  opens  them  under  0*1 
per  cent,  osmic  acid,  removes  as  much  of  the  albumen  as 
possible,  brings  the  yolk  for  twenty -four  hours  into  chromic 
acid  of  1  :  300  strength,  then  excises  the  blastoderm,  washes, 
puts  it  for  three  hours  into  a  mixture  of  glycerin,  alcohol, 
and  water  in  equal  parts,  and  lastly  into  90  per  cent, 
alcohol. 

See  also  previous  editions. 


Amphibia. 

600.  Preliminary. — In  order  to  prepare  ova  for  section- 
cutting,  it  is  essential  to  begin  by  removing  their  thick 
coats  of  albumen.  This  may  be  done  by  putting  them  for 
two  or  three  days  into  1  per  cent,  solution  of  chromic  acid, 
and  shaking  well ;  but  ova  thus  treated  are  very  brittle, 
and  do  not  afford  good  sections.  A  better  method  is  that 
described  by  WHITMAN  (Amer.  Natural.,  xxii,  1888,  p.  857), 
and  by  BLOCHMANN  (Zool.  Anz.,  1889,  p.  269).  WHITMAN 
puts  the  fixed  eggs  into  a  10  per  cent,  solution  of  sodium 
hypochlorite  diluted  with  5  to  6  volumes  of  water,  and 
leaves  them  there  till  they  can  be  shaken  free,  which 
happens  (for  Necturus)  in  a  few  minutes.  BLOCHMANN  takes 
eau  de  Javelle  (potassium  hypochlorite),  and  dilutes  it  with 
3  to  4  volumes  of  water,  and  agitates  the  eggs  previously 


342  CHAPTER  XXVI. 

fixed  with  solution  of  Flemming,  for  fifteen  to  thirty  minutes 
in  it.  Some  other  means  of  attaining  the  same  end  are 
given  in  the  following  paragraphs. 

A  great  difficulty  with  the  ova  of  Amphibia  lies  in  their 
becoming  extremely  brittle  on  imbedding  in  paraffin. 
CARNOY  and  LEBBUN  (La  Cellule,  xii,  1897,  p.  212)  fix 
ovaries  or  ovarian  ova  for  fifteen  minutes  to  three  quarters 
of  an  hour  in  Gilson's  mercuro-nitric  fluid,  §  70,  and  .pre- 
serve them  in  80  per  cent,  alcohol.  To  imbed,  they  are 
brought  for  a  quarter  of  an  hour  into  95  per  cent,  alcohol, 
five  minutes  in  absolute  alcohol,  then  into  a  mixture  of 
alcohol  and  chloroform  in  equal  parts,  and  as  soon  as  they 
sink  in  that  they  are  put  into  pure  chloroform.  Paraffin  is 
added  to  the  chloroform,  enough  to  about  double  the 
volume  of  the  whole,  and  the  whole  is  put  for  about  three 
hours  into  a  stove  at  35°  "C.  Lastly,  the  ova  are  put  for 
not  more  than  Jive  minutes  into  a  bath  of  pure  paraffin  at 
52°  C. ' 

See  also  MORGAN,  Devel.  of  the  Frog's  Egg,  New  York,  1897,  p.  171. 

601.  Axolotl. — The  ova   are  easier  to  prepare   than  those 
of    the   Anura,    because    the   yoke   is    separated    from    the 
albuminous  layer  by  a  wide  space  filled  with  a  liquid  that  is 
not  coagulated  by  reagents.      Put  the  eggs  for  a  few  hours 
into  picro-sulphuric  acid,  then  pierce  the  inner  chorion  with 
fine   scissors  or   needles,   and   gently   press    out   the    ovum. 
Harden  in  alcohol. 

602.  Triton  "(ScoTT  and  OSBORN,  Quart.  Journ.  Mic.  Soc., 
1879,  p.  449). — The  albumen  is  here  present  in  the  form  of 
several   concentric   coats,   which   are   very  delicate.      Incise 
each   of   them   separately   with    fine    scissors,   turn    out   the 
ovum,  and  fix  it.      Solution  of  Kleinenberg  is  the  reagent 
that  gives  the  best  results. 

HEKTWIG  (Jen.  Zeit.  f.  Naturw.,  1881-2,  p.  291)  puts  the 
eggs  into  a  mixture  of  equal  parts  of  2  per  cent,  acetic  acid 
and  0'5  per  cent,  chromic  acid.  After  ten  hours  he  incises 
the  membranes,  opening  one  end  of  the  inner  chorion,  and 
turns  out  the  embryos  and  brings  them  into  successive 
alcohols. 

BKAUS    (Jena  Zeit.,    xxix,   1894,    p.   443)    fixes    ova   to   a 


EMBRYOLOGICAL    METHODS.  343 

piece  of  liver  by  passing  an  entomological  pin  through  the 
albumen,  then  incises  the  albumen  and  turns  out  the  ova 
into  fixing  liquid. 

MICHAELIS  (Arch.  mik.  Anat.,  xlviii,  1896,  p.  528)  fixes 
ova  in  a  mixture  of  concentrated  sublimate  solution  and  con- 
centrated picric  acid,  twenty  parts  each,  glacial  acetic  acid  1, 
and  water  40,  but  removes  the  envelopes  before  bringing  into 
alcohol. 

603.  Salamandra  (RABL,  Morphol.  Jahrb.,   xii,  2,  1886,  p. 
252). — For  his  more  recent  methods  see  §  587. 

604.  Rana  (0.   HKRTWIG,  Jen.  Zeit.  f.  Naturw.,  xvi,    1883, 
p.    249). — The  ova    are   thrown    into    nearly  boiling    water 
(90°  to   96°  C.)   for  five  or  ten   minutes.      The    albuminous 
envelope  of  the  ovum  is  then  cut  open,  and  the  ovum  ex- 
tracted under  water.      The  ova  are  then   brought  into    0'5 
per  cent,  chromic  acid  for  not  more  than  twelve  hours,   or 
into   alcohol   of    70,   80,   and   90   per  cent.      Chromic   acid 
makes  ova  brittle  and  attacks   the  pigment,   whilst  alcohol 
preserves  it,  which  is  frequently  important  for  the  study  of 
the  germinal  layers. 

MOKGAN  (Amer.  Nat.,  xxv,  1891,  p.  759  ;  Journ.  Roy.  Mic. 
Soc.,  1892,  p.  284)  has  the  following.  During  the  periods 
in  which  it  is  difficult  or  impossible  to  remove  the  inner  jelly- 
membrane  the  eggs  can  be  freed  as  follows  : — Each  egg  is 
cut  out  with  scissors  from  the  general  jelly-mass,  and  put 
for  from  one  to  twelve  hours  into  saturated  solution  of  picric 
acid  in  35  per  cent,  alcohol  containing  ' '  the  same  amount  of 
sulphuric  acid  as  in  Kleinenberg's  solution."  Wash  for 
several  hours  in  several  changes  of  alcohol,  beginning  with 
35  per  cent.,  and  increasing  the  strength  gradually  up  to  70 
per  cent.  About  the  second  day  in  the  70  per  cent,  alcohol 
the  inner  membrane  begins  to  swell,  and  on  the  third  or 
fourth  day  may  be  pierced  by  a  needle,  and  the  egg  removed 
and  placed  in  80  per  cent,  alcohol  (see  also  WHITMAN,  Meth. 
of  Research,  p.  156 ;  and  SCHDLTZE,  Zeit.  f.  wiss.  ZooL,  xlv, 
1887,  p.  177). 

605.  Sulphate  of  Copper  Hardening  Liquid  (FoL,  Lehrbuch,  p.  106, 
after  REMAK  and  GOETTE)  ;  for  hardening  ova  of  Amphibia  : 


344  CHAPTER   XXVI. 

2  per  cent,  solution  of  sulphate  of  copper        .         .  50  c.c. 

Alcohol  of  25  per  cent. 50    „ 

Rectified  wood  vinegar  .         .         .         ...  35  drops. 


Pisces. 

606.  Teleostea  in  General. — The  ova  of  many  of  the  bony 
fishes  can  be  studied  by  transmitted  light  in  the  living  state  ; 
but  those  of  the  Salmonidse  must  be  hardened  and  removed 
from  their  envelopes  for  the  study  of  the  external  forms  of 
the  embryo. 

To  this  end  the  ova  may  be  put  for  a  few  minutes  into 
water  containing  1  to  2  per  cent,  of  acetic  acid,  and  thence 
into  1  per  cent,  chromic  acid.  After  three  days  the  capsule 
of  the  ovum  may  be  opened  at  the  side  opposite  to  the  em- 
bryo, and  be  removed  with  fine  forceps.  The  ovum  is  put 
for  twenty-four  hours  into  distilled  water,  and  then  into 
successive  alcohols.  Embryos  thus  prepared  show  no  defor- 
mation, and  their  histological  elements  are  fairly  well  pre- 
served. But  the  vitellus  rapidly  becomes  excessively  hard 
and  brittle,  so  as  greatly  to  interfere  with  section- cutting. 

The  following  processes  give  good  results  as  regards 
section-cutting. 

Put  the  ova  for  a  few  minutes  into  1  per  cent,  osmic  acid ; 
as  soon  as  they  have  taken  on  a  light  brown  colour  bring 
them  into  Miiller's  solution.  Open  them  therein  with  fine 
scissors — the  vitellus,  which  immediately  coagulates  on  con- 
tact with  air,  dissolves,  on  the  contrary,  in  Miiller's  solution 
— and  the  germ  and  cortical  layer  can  be  extracted  from  the 
capsule  of  the  ovum.  They  should  be  left  in  clean  Miiller's 
solution  for  a  few  days,  then  washed  with  water  for  twenty- 
four  hours,  and  brought  through  successive  alcohols. 

Another  method  (HENNEGUY)  is  as  follows  : — The  ova  are 
fixed  in  solution  of  Kleinenberg  containing  10  per  cent,  of 
acetic  acid.  After  ten  minutes  they  are  opened  in  water 
containing  10  per  cent,  of  acetic  acid,  which  dissolves  the 
vitellus.  The  embryos  are  put  for  a  few  hours  into  pure 
solution  of  Kleinenberg,  and  are  then  brought  through 
alcohol  of  gradually  increasing  strength. 

607.  KOLLMANN'S  Fixative  (KOLLMANN,  Arch.f.  Anat.  u.  Phys.,  1885, 
p.  296). 


EMBRYOLOGICAL    METHODS.  345 

Bichromate  of  potash    .         .         .         .         .5  per  100. 

Chromic  acid         .         .         .         .         .         .     2       „ 

Concentrated  nitric  acid        .         .         .         .     2       „ 

For  ova  of  Teleostea.  Fix  for  twelve  hours,  wash  with  water  for  twelve 
hours,  then  remove  the  chorion,  and  put  the  ova  into  70  per  cent,  alcohol. 

608.  RABL'S  Method  (see  §  587). 

609.  KOWALEWSKY'S  Method  (see  Zeit.f.  wiss.  ZooL,  xliii,  1886,  p.  434, 
or  Third  Edition). 

610.  Salmonidae. — HAKKISON  (Arch.  mik.  Anat.,  xlvi,  1895, 
p.  505)  fixes  embryos  of  Salmo  in  saturated  solution  of 
sublimate  in  5  per  cent,  acetic  acid. 

FELIX  (Anat.  Hefte,  1  Abth.,  viii,  1897,  p.  252)  fixes  the 
ova  for  three  quarters  of  an  hour  in  acetic  sublimate,  but 
dissected-out  embryos  in  liquid  of  Zenker,  the  vitellus  being 
removed  from  the  abdominal  cavity  with  a  brush. 

KOPSCH  (Arch.  mik.  Anat.,  li,  1897,  p.  184),  on  the  sug- 
gestion of  VIRCHOW,  fixes  the  embryos  for  five  or  ten  minutes 
in  a  mixture  of  1  part  of  chromic  acid  to  50  of  glacial  acetic 
acid  and  450  of  water,  then  removes  them  into  chromic  acid 
of  1  :  500,,  and  as  soon  as  may  be  removes  the  capsule  and 
yolk  under  salt  solution,  and  completes  the  hardening  in  the 
chromic  acid  or  saturated  sublimate  solution. 

Similarly  BEHRENS  (Anat.  Hefte,  H.  32,  1898,  p.  227  • 
Zeit.  f.  wifts.  Mik.,  xv,  1899,  p.  332).  He  leaves  the  ova  for 
about  an  hour  in  the  chromic  acid,  not  much  more  anyway ; 
he  opens  them  in  the  salt  solution  from  the  antipolar  side, 
and  frees  the  embryo  from  the  yolk  that  remains  by  blowing 
the  latter  away  with  a  fine-pointed  glass  tube  ;  after  which 
the  embryo  can  be  easily  detached  from  the  capsule.  It  is 
then  removed  for  about  three  hours  into  a  mixture  of  1  part 
saturated  picric  acid  solution,  1  part  saturated  sublimate 
solution,  and  2  parts  distilled  water,  after  which  it  is  treated 
in  the  usual  way  with  iodine  and  successive  alcohols. 

611.  RABL-RUCKHABD'S  Method  for  Salmonidse  (Arch.  f.  Anat.  u. 
Entw.,  1882,  p.  118).— Fix  in  10  per  cent,  nitric  acid  for  fifteen  minutes. 
Remove  the  membranes  to  avoid  deformation  of  the  embryos,  and  put  the 
ova  back  into  the  acid  for  an  hour.  Wash  out  in  1  to  2  per  cent,  solution 
of  alum  for  an  hour,  and  harden  in  alcohol. 

Modification  of  this  method  by  GOBONOWITSCH  (see  Morph.  Jahrb.,  x, 
1884,  p.  381). 


346  CHAPTER  XXVI. 

612,  Selachii— SCHMIDT'S  Method  (see  Zeit.f.  wiss.  Mik.,  xv,  3,  1899, 
p.  332). — Fix  in  acetic  sublimate. 

613.  Pelagic  Fish.  Ova— WHITMAN'S  Method  (Amer.  Natural,  xvii, 
1883,  pp.  1204-5 ;  Journ.  Boy.  Mic.  Soc.  [N.  S.],  iii,  1883,  p.  912  ;  and 
Methods  of  Research,  etc.,  p.  152). — Fix  by  treatment  first  for  five  to  ten 
minutes  with  a  mixture  of  equal  parts  of  sea  water  and  |  per  cent,  osmic 
acid  solution,  and  then  for  one  or  two  days  with  a  solution  (due  to  Eisig) 
of  equal  parts  of  0'25  per  cent,  platinum  chloride  and  1  per  cent,  chromic 
acid.    Prick  the  membrane  before  transferring  to  alcohol.    See  also  AGASSIZ 
and  WHITMAN,  in  Proc.  Amer.  Acad.  Arts  and  Sciences,  xx,  1884 ;  and 
COLLINGE,  Ann.  and  Mag.  Nat.  Hist.,  x,  1892,  p.  228 ;  Journ.  Roy.  Mic. 
Soc.,  1892,  p.  883. 

RAFFAELE  (Mitth.  Zool.  Stat.  Neapel,  xii,  1895,  p.  169)  fixes  chiefly  with 
liquid  of  Hermann  (1  to  2  days),  or  with  mixture  of  Mingazzini  (absolute 
alcohol  1,  acetic  acid  1,  saturated  sublimate  solution  in  water  2). 

614.  Amphioxus. — SOBOTTA   (Arch.   mik.  Anat.,  1,   1897,  p. 
20)    advises    fixing    for    twenty-four     hours    in    liquid     of 
Flemming. 

Tunicata. 

615.  Ova.— DAVIDOFF  (Mitth.  Zool.  Stat.  Neapel,  ix,  1,  1889, 
p.  118)  fixes  the  ova  of  Distaplia  with  a  mixture  of  3  parts 
of  saturated  solution  of  corrosive  sublimate  and  1  of  glacial 
acetic  acid.      The  ova  to  remain  in  it  for  from  half  an  hour 
to  an  hour,  and  be  then  washed  for  a  few  minutes  in  water 
and  brought  through  successive  alcohols.      Another  reagent, 
almost  as  good,  consists  of  3  parts   of  saturated  solution  of 
picric  acid  and  1  of  glacial  acetic  acid,  the  objects  to  remain 
in  it  for  three  to  four  hours,  and  then  be  brought  into  70  per 
cent,  alcohol. 

CASTLE  (Bull.  Mus.  Harvard  Coll.,  xxvii,  1896,  p.  213)  advises  for  ova 
of  Ciona  liquid  of  Perenyi  for  twenty  minutes,  followed  by  70  per  cent, 
alcohol  for  twenty-four  hours,  and  for  the  larvae  picro-nitric  acid. 

616.  Test-Cells  of  Ascidians  (MOEGAN,  Journ.  of  Morphol.,  iv,  1890, 
p.  195). — Tease  fresh  ovaries  in  very  weak  osmic  acid,  wash  in  distilled 
water,  treat  for  half  an  hour  with  1  per  cent,  silver  nitrate,  wash  for  half 
an  hour  in  2  per  cent,  acetic  acid,  and  reduce  in  sunlight.  Imbed  in  paraffin. 
By  this  process  the  limits  of  the  follicle  cells  are  demonstrated. 

,  617.  Buds. — PIZON  (Ann.  Sc.  Nat.,  xix,  1893,  p.  5)  studies 
the  gemmation  of  the  composite  Ascidians  either  on  entire 


EMBRYOLOGIOAL    METHODS.  347 

conns,  which  he  first  bleaches  with  peroxide  of  hydrogen 
(which  acts  less  brutally  than  eau  de  Javelle,  but  the  bubbles 
that  arise  must  be  removed  with  an  air  pump),  and  then 
stains  ;  or  by  making  sections,  after  anaesthetising  the  colonies 
with  cocain  of  1  :  1000,  fixing  in  glacial  acetic  acid  or  picro- 
sulphuric  or  liquid  of  Flemming,  and  staining  in  toto  with 
borax  carmine  or  alum  carmine,  or  with  a  strong  solution  of 
methylen  blue  in  alcohol  of  90  or  100  per  cent,  (after  BERNARD, 
ibid.,  ix,  1890,  p.  97). 

HITTER  (Joum.  of  Morph.,  xii,   1896,  p.  150)  recommends 
for  fixing  Perophora  and  Goodsiria  picro-sulphuric  acid. 


Bryozoa. 

618.  Statoblasts. — BRA  EM  (Bibl.  ZooL,  Chun  and  Leuckart, 
6  Heft,  1890,  p.  95)  fixes  statoblasts  of  Cristatella  with  hot 
concentrated  solution  of  sublimate  for  ten  minutes,  brings 
them  into  water  and  there  incises  them  with  a  razor,  and 
after  half  an  hour  passes  them  gradually  into  alcohol.  He 
stains  with  picro-carmine. 


Mollusca. 

619.  Cephalopoda  (Ussow,  Arch,  de  BioL,  ii,  1881,  p.  582). 
— Segmenting  ova  are  placed,  without  removal  of  the  mem- 
branes, in  2  per  cent,  solution  of  chromic  acid  for  two 
minutes,  and  then  in  distilled  water,  to  which  a  little  acetic 
acid  (one  drop  to  a  watch-glassful)  has  been  added,  for  two 
minutes.  If  an  incision  be  now  made  into  the  egg-mem- 
brane the  yolk  flows  away  and  the  blastoderm  remains ;  if 
any  yolk  still  cling  to  it,  it  may  be  removed  by  pouring 
away  the  water  and  adding  more. 

WATASE  (Joum.  of  MorphoL,  iv,  1891,  p.  249;  Journ. 
Roy.  Mic.  Soc.,  1892,  p.  152)  kills  the  ova  in  the  macerating 
mixture  of  the  Hertwigs  (§  538),  and  as  soon  as  the 
blastoderm  turns  white  and  opaque  removes  it  under  dilute 
glycerin.  Treatment  with  liquid  of  Perenyi  is  recommended 
for  surface  views. 

VIALLETON  (Ann.  Sc.  Nat.,  vi,  1887,  p.  168)  brings 
ovarian  ova  of  Sepia  into  a  freshly-prepared  mixture  of 


348  CHAPTER    XXVI. 

picro-sulphuric  acid  and  2  per  cent,  solution  of  bichromate  of 
potash  in  equal  parts,,  and  after  one  or  two  minutes  incises 
them  in  the  equator,  fixes  for  an  hour  and  a  half  in  picro- 
sulphuric  acid  the  halves  that  contain  the  formative  vitellus, 
separates  this  from  the  nutritive  vitellus  with  a  spatula, 
spreads  it  out,  and  hardens  it  in  alcohol  of  70  to  90  per 
cent.  He  fixes  entire  ova  in  liquid  of  Flemming  or  osmic 
acid. 

KOESCHELT  (Festschrift  Leuckart,  Leipzig,  1892,  p.  348) 
fixes  advanced  embryos  of  Loligo  in  liquid  of  Flemming, 
sublimate,  picro-sulphuric  acid,  or  0'2  per  cent,  chromic  acid. 
This  last  is  specially  good  for  young  embryos  if  it  is  washed 
out  with  many  changes  of  picric  acid. 

620.  Gastropoda  (HENNEGUY). — Ova  of  Helix  may  be  fixed 
for  from  four  to  six  hours  in  Mayer's  picro-nitric  acid. 
The  carbonate  of  lime  that  encrusts  the  external  membrane 
is  thus  dissolved,  and  the  albuminous  coat  of  the  egg  is 
coagulated.  The  egg  is  opened  with  needles,  the  albumen 
comes  away  in  bits,  and  the  embryo  can  be  removed.  Treat 
with  successive  alcohols,  and  imbed  in  paraffin. 

Miss  A.  HENCHMAN  (Bull.  Mus.  Comp.  ZooL,  Harvard,  xx, 
1890,  p.  171;  Journ.  Roy.  Mic.  Soc.,  1891,  p.  274;  Zeit.  f. 
wiss.  Mik.,  viii,  2,  1891,  p.  216)  fixes  ova  of  Limax  with 
0'33  per  cent,  chromic  acid,  or  with  liquid  of  Perenyi.  It 
is  best  to  remove  only  the  outer  envelope  before  putting 
into  the  chromic  acid,  the  inner  membrane  being  removed 
after  two  or  three  minutes  therein.  Where  Perenyi  is  used 
the  membranes  must  be  removed  first,  as  the  albumen  will 
else  coagulate  in  such  a  way  as  to  prevent  the  removal  of 
the  embryos.  ,For  the  manner  of  obtaining  the  ova,  see 
previous  editions. 

MEISKNHKIMER  (Zeit.  wiss.  ZooL,  Ixii,  1896,  p.  417)  dissects 
out  the  embryos  of  Limax  and  fixes  them  with  picro-sulphuric 
acid  or  concentrated  sublimate.  Advanced  embryos  are 
first  got  into  extension  by  means  of  2  per  cent,  cocain,  or 
are  rapidly  killed  with  hot  sublimate. 

SCHMIDT  (Entw.  Pulmonaten,  Dorpat,  1891,  p.  4)  fixes  the 
ova  in  toto  with  concentrated  sublimate,  and  dissects  them 
out  afterwards. 

Similarly  KOFOID   (Bull.  Mus.  Harvard  Coll.,  xxvii,  1895, 


E.MBRYOLOGICAL    METHODS.  349 

p.  35).  Or,  preferably,  the  ova  are  put  into  salt  solution, 
the  shell  removed,  the  albumen  removed  with  a  pipette  full 
of  salt  solution,  which  dissolves  it ;  the  ova  are  then  fixed 
for  one  minute  in  Pol's  modification  of  liquid  of  Flemming, 
and  brought  direct  into  Orth's  picro-lithium-carmine. 

See  also  WASHBUBN,  Amer.  Nat.,  xxviii,  1894,  p.  528  (liquid  of  Flem- 
ming or  0*3  per  cent,  chromic  acid,  or  1  per  cent,  osmic  acid,  followed  by 
liquid  of  Merkel). 

CONKLIN  (Journ.  of  Morph.,  xiii,  1897,  p.  7)  fixes  ova  of  Crepidula  for 
fifteen  to  thirty  minutes  in  picro-sulphuric  acid,  and  stains  with  dilute 
acidified  haematoxylin  of  Delafield. 

KOSTANECKI  and  WIERZEJSKI  (Arch.  f.  mik.  Anat.,  xlvii, 
1896,  p.  313)  fix  the  spawn  of  Physa  fontinalis  either  in  1J 
to  2  per  cent,  nitric  acid,  or  in  "  sublimate  and  3  per  cent, 
nitric  acid  in  the  proportion  of  2  :  1,"  and  bring  through 
successive  alcohols.  They  imbed  entire  ova  in  paraffin,  but 
isolated  embryos  in  celloidin. 

CARAZZI  (Anat.  Anz.,  xvii,  1900,  p.  78)  for  Aplysia  takes 
5  per  cent,  sublimate  with  2*5  per  cent,  glacial  acetic  acid. 

621.  CHITON,  see  METCALF,  Stud.  Biol.  Lab.  Johns  Hopkins  Univ.,  v, 
1893,  p.  251,  or  Joum.  Roy.  Mic.  Soc.,  1894,  p.  531. 

622.  Lamellibranchiata. — STAUP PACKER  (Jena  Zeit.,  xxviii, 
1893,  p.  196)  fixes  embryos  of  Cyclas  in  sublimate,  stains 
with  haemalum,  and  cuts  in  paraffin. 

LILLIE  (Journ.  of  Morph.,  x,  1895,  p.  7)  fixes  ova  of  Unio 
for  ten  to  twenty  minutes  in  liquid  of  Perenyi,  and  preserves 
them  in  70  per  cent,  alcohol,  or  advanced  embryos  with  liquid 
of  Merkel  or  sublimate,  larvae  with  0'05  to  O'l  per  cent,  osmic 
acid,  preserving  them  in  glycerin.  Glochidia  may  be  cut 
with  the  shell  in  paraffin  of  58°  melting-point ;  they  may  be 
anaesthetised  with  chloral  hydrate  before  fixing. 


Arthropoda. 

623.  Fixation  of  Ova. — In  most  cases  the  ova  of  Arthropods 
are  fixed  by  heat  in  a  more  satisfactory  way  than  by  any 
other  means.  This  may  be  followed  either  by  alcohol  or 
some  watery  hardening  agent.  If  it  be  desired  to  avoid 
heating,  picro-sulphuric  acid  may  be  tried. 


350  CHAPTER    XXVI. 

624.  Removal  of  Membranes.— This  is  frequently  very  diffi- 
cult, and  it  may  often  be  advisable  not  to  attempt  to  remove 
them,  but  to  soften  them  with  eau  de  Javelle  or  eau  de  Labar- 
raque  (see  §  556). 

MORGAN  (Amer.  Natural.,  xxii,  1888,  p.  357  ;  Zeit.  f.  wiss. 
Nik.,  vi,  1,  1889,  p.  69)  recommends  (for  the  ova  of  Peri- 
planeta)  eau  de  Labarraque  diluted  with  five  to  eight  volumes 
of  water,  and  slightly  warmed.  Thus  used  it  will  soften  the 
chitin  membranes  sufficiently  in  thirty  to  sixty  minutes,  if 
employed  before  fixing.  Fixed  ova  take  longer.  The  fluid 
must,  of  course,  not  be  allowed  to  penetrate  into  the  interior 
of  the  ovum. 


625.  HENKING' s  Methods  (Zeit.  f.  wiss.  Mik.,  viii,  2,  1891, 
p.  156). — HENKING  generally  kills  ova  by  plunging  them  into 
hot  water,  or  by  pouring  hot  water  on  to  them  in  a  watch 
glass,  and  then  removing  into  70  per  cent,  alcohol.  But  he 
finds  that  the  preservation  of  structures  by  this  method  is 
far  from  being  perfectly  satisfactory,  cell  contours  being  not 
at  all  sharply  brought  out  by  it,  and  achromatic  cell  structures 
being  but  imperfectly  preserved.  He  finds  that  in  some  cases 
ova  may  be  fixed  with  liquid  of  Flemming,  which  then  gives 
incomparably  better  results  in  these  respects.  Suitable  ova 
may  be  put  into  liquid  of  Flemming  (HENKING  does  not  say 
which  formula)  for  half  an  hour,  then  for  two  hours  into  the 
same  diluted  with  three  volumes  of  water,  then  treated  with 
alcohol  as  usual.  BOVEKI'S  picro-acetic  acid  was  found  not 
to  penetrate  the  membranes.  -I 

HENKING  thinks  that  eau  de  Javelle  for  softening  mem- 
branes is  best  avoided.  They  should  either  be  dissected 
away  or  left  in  situ,  and  cut  with  the  rest  of  the  egg,  accord- 
ing to  the  nature  of  the  case.  The  great  obstacle  to  section- 
cutting  is  the  brittleness  of  the  yolk.  This  difficulty  may  be 
overcome  as  follows  : — After  fixing  and  treating  with  alcohol, 
prick  the  chorion  and  stain  with  borax- carmine.  Put  the 
stained  ova  for  twelve  hours  into  a  mixture  containing 
20  c.c.  of  70  per  cent,  alcohol,  one  drop  of  concentrated 
hydrochloric  acid,  and  a  knife  pointful  of  pepsin  (it  is  not 
necessary  that  all  the  pepsin  should  be  dissolved).  The  ova 
may  then  be  treated  with  alcohol,  oil  of  bergamot,  and 


EMBRYOLOGICAL    METHODS.  351 

paraffin,  and  (with  some  exceptions,  amongst  which  is  Bombyx 
mori)  will  be  found  to  cut  without  crumbling. 

The  contents  of  fresh  ova  may  conveniently  be  studied  by 
means  of  the  following  fluid : 

Distilled  water  .          .          .          .      80  c.c. 
Glycerin    .  .  .         -..  16 

Formic  acid        .  .          .  .        3    „ 

1  per  cent,  osmic  acid.         ..         '.        1    „ 
Dahlia       .  .  .  .          ,.      0'04  grm. 

The  eggs  are  simply  teased  in  a  drop  of  the  liquid,  and  a 
cover-glass  put  on.  If  it  be  desired  to  preserve  the  prepara- 
tion, nothing  more  is  necessary  than  to  lute  the  cover-glass. 

626.  Diptera   (HENKING,   Zeit.  f.    wiss.   Zool,  xlvi,    1888, 
p.  289  ;  Zeit.  /.  wiss.  Mik.,  1889,  p.  59).— Ova  still  contained 
within  the  fly  may  be  fixed  by  plunging  the  animal  for  some 
time  into   boiling  water,  then  dissecting   out  and   bringing 
them  into  70  per  cent,  alcohol.     Laid  eggs  may  have  boiling 
water  poured  over  them,  or  be  put  into  solution  of  Flemming 
in  a  test-tube  which  is  plunged  into  boiling  water  until  the 
eggs  begin  to  darken   (about   a  minute).      Cold  solution  of 
Flemming  easily  causes  a  certain  vacuolisation  of  the  contents 
of  the  ova.    Open  the  ova  at  the  larger  end,  stain  with  borax- 
carmine  for  fifteen  to  thirty  hours,  and  cut  in  paraffin. 

See  also  (for  Chironomus)  RITTER,  Zeit.  f.  wiss.  Zool.,  i, 
1890,  p.  408;  Zeit.f.  wiss.  Mik.,  viii,  1,  1891,  p.  87  (strings 
of  ova  fixed  with  hot  30  per  cent,  alcohol  containing  a  little 
sublimate,  and  stained  in  the  mass  by  immersion  for  several 
days  in  picro-carmine). 

BRUEL  (Zool.  Jahrb.,  Abth.  Morph.,  x,  1897,  p.  569)  fixes 
larvae  and  pupae  in  absolute  alcohol  heated  to  70°  to  75°  C., 
and  containing  "  a  little  "  sublimate.  See  also  VaN  REES, 
ibid.,  iii,  1888,  p.  10. 

BENGTSSON  (Handl.  Fysiogr.  8aellsk.  Lund.,  viii,  1897)  finds 
hot  alcoholic  solution  of  sublimate  (Frenzel's,  §  70)  the  best 
fixative  for  larvae  of  Phalacrocera.  He  could  not  succeed  in 
softening  the  chitin  with  eau  de  Javelle. 

627.  Lepidoptera  (BOBRETZKY,  Zeit.f.  wiss.  Zool.,  1879,  p. 
198). — Ova  are  slightly  warmed  in  water  and  put  for  sixteen 
to  twenty  hours  in  0*5  per  cent,  chromic  acid.      The  mem- 


352  CHAPTER    XXVI. 

branes  can  then  be  removed,  and  the  ova  brought  for  a  few 
hours  into  absolute  alcohol,  stained  with  carmine,  and  cut. 

628.  Blattida   (PATTEN,   Quart.  Journ.   Mic.   Sci.,   1884,  p. 
549). — The  ova  or  larvae  are  placed  in  cold  water,  which  is 
gradually  raised  to  80°  C.      You  leave  off  heating  as  soon  as 
the  ova  have  become  hard  and  white.       Pass  very  gradually 
through  successive  alcohols,  beginning  with  20  per  cent. 

WHEELED  (Journ.  of  Morph.,  iii,  1889,  p.  292;  Journ.  Roy. 
Mic.  Soc.,  1890,  p.  250)  dissects  out  ovarian  ova  in  salt  solu- 
tion and  fixes  in  liquid  of  Perenyi  (fifteen  minutes),  then 
treats  with  alcohol,  and  stains  with  borax-carmine.  Laid 
eggs  may  be  killed  by  Patten's  method.  After  heating,  the 
two  lips  of  the  crista  of  the  capsule  may  be  separated  with 
fine  forceps  and  pieces  of  the  walls  torn  away,  and  the  eggs 
pushed  out  of  the  compartments  formed  by  their  choria  and 
hardened  as  desired.  Good  results  are  also  obtained  by 
heating  to  80°  C.  for  ten  minutes  in  liquid  of  Kleinenberg, 
and  preserving  in  70  per  cent,  alcohol.  This  causes  the  en- 
velopes to  dilate  and  stand  off  from  the  surface  of  the  egg, 
so  that  they  can  easily  be  dissected  away. 

HEYMONS  (Zeit.f.  wiss.  Zool.,  liii,  1892,  p.  434 ;  Zeit.f.  wiss. 
Mik.,  ix,  3,  1893,  p.  343),  for  young  embryos,  incises  the 
cocoon  at  the  end  by  which  it  adheres  in  the  body  of  the 
mother,  brings  it  for  two  minutes  into  water  heated  to  90°  C., 
and  opens  in  Flemming,  in  which  the  embryo  is  dissected 
out. 

629.  Coleoptera. — LECAILLON   (Arch.  Anat.  Micr.,  1,   1897, 
p.  208)  fixes  ova  for  twenty -four  hours  in  liquid  of  Zenker 
warmed  to  40°  G. 

630.  Phalangida  (HENKING,  Zeit.f.  wiss.  ZooL,  xlv,  1886,  p.  86  ;  Zeit. 
f.  wiss.  l\fik.,iu,  4, 1886,  p.  470). — Fix  with  boiling  water  or  Flemniing. 
Preserve  the  ova  in  90  per  cent,  alcohol.  To  open  the  chorion,  bring  them 
back  into  70  per  cent,  alcohol,  which  causes  them  to  swell  up  so  that  the 
chorion  can  easily  be  pierced  with  needles,  and  the  ovum  turned  out. 

631.  Araneida. — KISHINOUYE  (Journ.  Coll.  Sci.  Imp.  Univ. 
Japan,  iv,  1891,  p.  55 ;  Zeit.  f.  wiss.  Mik.,  ix,  2,  1892,  p.  215) 
fixes  in  water  warmed  to  70°  or  80°  C.,  puts  into  70  per  cent, 
alcohol,  and  after  twenty-four  hours  therein  pierces  the  me  in- 


EMBRYOLOGTCAL    METHODS.  353 

branes    and   passes    through    stronger   alcohol.      Stain  with 
alcoholic  cochineal  or  picro-carmine,  and  imbed  in  paraffin. 

See  also  LOCY,  Bull.  Mus.  Comp.  Zool,  Harvard,  xii,  3,  1886;  Zeit.f. 
wiss.  Mik.,  iii,  2,  1886,  p.  242.  Fix  by  hot  water.  The  liquid  of  Perenyi 
may  also  be  used ;  it  has  the  advantage  of  not  making  the  yolk  so  granular. 

632.  Scorpionidea. — BRAUEK  (Zeit.f.  wiss.  Zool.,  Ivii,  1894, 
p.  405)  removes  ovarian  tubes  from  living  Euscorpius,  and 
fixes  the  small  ova  for  half  an  hour  to  an  hour  in  liquid  of 
Flemming,  then  dissects  out  the  ova.  He  puts  tubes  with 
more  advanced  embryos  for  twenty-four  hours  into  one  fifth 
per  cent,  chromic  acid,  or  for  a  minute  to  a  minute  and  a 
half  into  almost  boiling  water,  and  then  for  two  to  six  hours 
into  chromo-acetic  acid,  or  for  ten  to  twenty  minutes  into 
liquid  of  Flemming.  The  chromic  acid  material  is  only  good 
for  surface  views. 

633.  Tardigrada. — ERLAXGEE  (Morph.  Jahrb.,  xxii,  1895,  p.  493)  fixes 
the  ova  (and  the  animals  themselves)  in  liquid  of  Flemming  or  in  picro- 
sulphuric  acid  containing  1  drop  of  1  per  cent,  osmic  acid  per  c.c.,  or  in 
concentrated  sublimate  solution  with  20  per  cent,  acetic  acid.  The  two  first 
require  to  be  bleached  with  warm  peroxide  of  hydrogen. 

634.  Limulus. — KINGSLEY  (Journ.  of  Morph.,  vii,  1892,  p.  38) 
warms  the  ova  in  sea  water  to  70°  or  75°  C.,  and  then  brings 
them  into  alcohol  of  30  to  70  per  cent.      For  early  stages  he 
marks  the  position  of  the  embryo  with  Indian  ink   on  the 
chorion  ;  the  mark  resists  alcohol.      Early  stages  should  be 
imbedded  in  celloidin,  later  ones  in  paraffin.      For  the  pre- 
paration of  surface  views  see  op.  cit.,  xii,  1896,  p.  23. 

Similarly  KISHINOUYE,  Joum.  Coll.  Sc.  Japan,  v,  1893, 
p.  56. 

635.  Astacus  (REICHENBACH,  Abk.  Senckenberg.   Ges.  Frank- 
furt, xiv,  1886,  p.  2;  Zeit.f.  wiss.  Mik.,  1886,  p.  400).— Fix 
in  water  gradually  warmed  to  60°  or  70°  C.  (if  the  chorion 
should  burst,  that  is  no  evil),  harden  for  twenty-four  hours 
in    1  to  2  per  cent,  bichromate  of  potash   or  0'5  per  cent, 
chromic  acid,  wash  out  for  the  same  time  in  running  water, 
and  bring  into   alcohol.      Remove  the  chorion,  and  remove 
the  embryo  from  the  yolk  by  means  of  a  sharp  knife. 

23 


354  CHAPTEE   XXVI. 

636.  Amphipoda. — DELLA    VALLE    (Fauna    u.    Flora    Golf. 
Neapel,  xx,  Monog.,  1893,  p.  170)  puts  ova  of   Orchestia  by 
means  of  a  pipette  into  boiling,  cold-saturated  sublimate  solu- 
tion, removes  them  instantly  into  sea  water,  and  thence  into 
weak  alcohol.    If  the  chorion  does  not  burst  of  itself  it  must 
be  pricked  with  a  needle. 

637.  Copepoda. — HAECKER  (Arch.  f.  mik.  Anat.,  xlix,  1897, 
p.  35)  fixes  ovisacs  of  Cyclops  for  ten  minutes  in  VOM  RATE'S 
picro  -  platin-osmic  mixture . 

638.  Isopoda. — McMuEEicn   (Journ.    of   Morph.,    xi,    1895, 
p.  65)  fixes  ova  of  Jaera  in  a  saturated  solution  of  picric  acid 
in  70  per  cent,  alcohol,  to  which  has  been  added  2  per  cent, 
of   concentrated   sulphuric  acid.       If   the  yolk  is  brittle  he 
imbeds  in  celloidin ;  if  not,  in  paraffin. 


Vermes. 

639,  Polychaeta.— WILSON  (Journ.  of  Morph.,  vi;  1892, 
p.  373)  stains  living  embryos  of  Nereis  with  methylen  blue 
in  sea  water.  He  fixes  generally  for  ten  to  thirty  minutes 
with  liquid  of  Flemming  or  Perenyi  or  Lang's  sublimate ; 
picro-sulphuric  acid  is  not  good. 

WISTINGHAUSEN  (Mitth.  Zool.  8 tat.  Neapel,  x,  1891,  p.  47) 
got  his  best  results  with  advanced  embryos  by  fixing  for  an 
hour  in  a  mixture  of  1  part  1  per  cent,  osmic  acid,  25  parts 
1  per  cent,  chromic  acid,  5  parts  acetic  acid,  glacial,  and  70 
parts  water,  washing  out  for  twenty-four  hours  in  water  and 
bringing  into  50  per  cent,  alcohol  for  three  hours,  then  into 
70  per  cent. 

KOESCHELT  (Zeit.  f.  wiss.  Zool.,  Ix,  1895,  p.  545)  found 
picro-acetic  acid  of  Boveri  the  best  thing  for  ova  of  Ophryo- 
trocha  :  time,  three  to  four  hours,  then  70  per  cent,  alcohol. 

KLEINENBEEG  (ibid.,  xliv,  1886,  p.  25)  fixes  larvae  of  Lopadorhynchus 
with  his  picro-sulphuric  acid,  brings  them  into  70  per  cent,  alcohol,  then 
90  per  cent.,  and  stains  with  borax  carmine.  He  also  macerates  them  for 
one  or  two  hours  in  dilute  picro-sulphuric  acid,  and  then  for  twenty-four 
hours  in  Beale's  carmine. 


EMBRYOLOGICAL    METHODS.  355 

EISIG  (Mitth.  ZooL  Stat.  Neapel,  xiii,  1898,  p.  89)  fixes 
ova  and  larvae  of  Capitella  with  a  freshly  prepared  mixture 
of  3  parts  5  per  cent,  solution  of  sublimate  in  sea  water  and 
1  part  glacial  acetic  acid ;  if  necessary  they  may  be  first 
narcotised  with  2  per  cent,  solution  of  cocaine  in  sea  water 
(the  precipitate  produced  by  the  sublimate  dissolves  after- 
wards in  alcohol).  Then  successive  alcohols.  He  stains  with 
haemacalcium  (with  5  per  cent,  acetic  acid  instead  of  2  per 
cent.),  and  washes  out  by  prolonged  treatment  with  alcohol 
containing  2  per  cent,  of  nitrate  of  aluminium. 

640.  Rotatoria.— JENNINGS  (Bull.  Mus.  Harvard  Coll.,  xxx, 
1896,  p.  101)  finds  the  best  fixative  for  pregnant  females  is 
the  strong  liquid  of  Flemming,  but  the  ova  must  then  be 
bleached  with  chlorate  of  potash  (§  575).      He  preserves  in 
80  per  cent,  alcohol,  or  in  a  mixture  of  equal  parts  of  alcohol, 
water,  and   glycerin.      He   dissects   out   the  ova  under  the 
microscope,  and  brings   them  into   glycerin   under  a  cover 
supported  on  capillary  glass  tubes  so  as  to  allow  of  rolling 
the  eggs. 

641.  Turbellaria.— GARDINER  (Journ.  of  Norph.,   xi,   1895, 
p.   158)   finds  the  best  fixative  for  ova  of  Polychoerus  is  a 
mixture  of  equal  parts  of  absolute  alcohol  and  glacial  acetic 
acid. 

Method  of  IIJIMA  (Zeit.  f.  iviss.  ZooL,  xl,  1884,  p.  359).— 
The  capsule  containing  the  ova  (of  fresh-water  Planaria)  is 
opened  with  needles  on  a  slide,  in  a  drop  of  2  per  cent,  nitric 
acid.  The  ova  are  extracted  and  covered  (the  cover  being 
supported  by  paper  or  by  wax  feet).  After  half  an  hour 
they  are  treated  with  successive  alcohols  under  the  cover, 
and  finally  mounted  in  glycerin.  For  sections,  the  whole  of 
the  contents  of  a  capsule  is  hardened  in  the  mass  in  1  per 
cent,  chromic  acid  and  cut  together. 

642.  Cestoda  (v.  BENEDEN,  Arch,  de  EioL,  ii,  1881,  p.  187).— 
Ova  of  Tdenia  in  which   a  chitinous  membrane  has  formed 
around  the  embryo  are  impervious  to  reagents.      They  may 
be  put  on  a  slide  with  a  drop  of  some  liquid  and  covered. 
Then,  by  withdrawing  the  liquid  by  means  of  blotting-paper, 
the  cover  may  be  made  to  gradually  press  on  them  so  as  to 
burst  the  membranes,  and  the  embryo  may  then  be  treated 
with  the  usual  reagents. 


356  CHAPTER    XXVI. 

643.  Trematoda. — COE  (Zool.  Jahrb.,  Abth.  Morph.,  ix,  1896, 
pp.  563,  566)  fixes   the   Miracidia  of   Distomum  for  general 
purposes  with  the  usual  fixatives ;  but  for  the  special  study 
of  the  excretory  system  he  kills  them  with  osmic  acid,  rinses 
with  distilled  water,  and  puts  them  for  a  couple  of  days  into 
£  per  cent,  solution  of  silver  nitrate. 

644.  Nematoda. — BOVERI    (Jena.   Zeit.,   xxi,    1887,    p.   423) 
fixes  the  ova  of  Ascaris  in  his  picro-acetic  acid  (after  which 
treatment  I  should  say  you  may  easily  see  anything  you  like 
to  imagine  in  them). 

ZUR  STEASSEN  (Arch.  Entwickelungsmecli.,  iii,  1896,  p.  29) 
fixes  them  for  twenty- four  hours  in  a  mixture  of  4  parts 
96  per  cent,  alcohol  and  1  part  acetic  acid  (much  too  long, 
I  should  say),  brings  them  into  pure  alcohol,  stains  with 
hydrochloric  acid  carmine,  and  brings  them  gradually  into 
glycerin. 

Similarly  ZOJA  (Arch.  f.  m%k.  Anat.,  xlvii,  1896,  p.  218) 
and  ERLANGER  (ibid.,  xlix,  1897,  p.  309).  Zoja  stained  with 
Bismarck  brown  and  examined  in  dilute  glycerin ;  Erlanger 
made  paraffin  sections  and  stained  with  iron  hsematoxylin. 

KOSTANECKI  and  SIEDLECKI  (ibid.,  xlviii,  1896,  p.  184) 
employed  concentrated  sublimate  solution,  or  3  per  cent, 
nitric  acid,  or  mixtures  of  these  two,  for  ovarian  ova. 

I  fancy  the  best  fixative  for  ova  furnished  with  their  cap- 
sules will  be  found  to  be  that  of  G-ILSON  CARNOY-LEBRUN 
(§  84)  ;  see  CARNOY  &  LEBRON,  La  Cellule,  xiii,  1897,  p.  68. 
After  fixation  the  ova  are  carefully  brought  into  80  per  cent, 
alcohol,  in  which  they  are  preserved.  Imbedding  should  be 
carefully  done,  as  recommended  for  the  ova  of  Amphibia 
(§  600),  but  they  ought  not  to  remain  in  the  pure  paraffin 
for  more  than  a  minute  to  a  minute  and  a  half.  But  these 
authors  prefer  the  celloidin  method.  At  least  six  weeks' 
soaking  in  the  different  strengths  of  celloidin  will  be  neces- 
sary to  ensure  penetration.  They  stain  with  iron  haema- 
toxylin. 

Echinodermata,  Coelenterata,  and  Porifera. 

See  the  paragraphs  treating  of  these  groups  in  the  chapter 
on  "Zoological  Methods." 


CHAPTER  XXVII. 

CYTOLOGICAL    METHODS. 

645.  Study  of  Living  Cells. — In  the  young  larvae  of  Am- 
phibia, both  Anura  and  Urodela,  the  gills  and  caudal  ' '  fin," 
and  sometimes  other  regions,  may  be  conveniently  studied  in 
the  living  state. 

The  larvae  may  be  fixed  in  a  suitable  cell,  or  wrapped  in 
moist  blotting-paper,  or  may  be  curarised ;  or  the  tail  may 
be  excised.  (It  is  preferable  to  cut  through  the  larva  close 
in  front  of  the  hind  limbs.) 

In  the  living  animal  the  epithelial  cells  and  nuclei  (in  the 
state  of  repose)  are  so  transparent  as  to  be  hardly  visible  in 
the  natural  state.  They  may,  however,  be  brought  out  by 
curarising  the  larva ;  or,  still  better,  by  placing  the  cura- 
rised larva  for  half  an  hour  in  1  per  cent,  chloride  of  sodium 
solution.  Normal  larvae  may  be  used  for  the  study  of  the 
active  state  of  the  nucleus,  but  much  time  is  saved  by  using 
curare. 

Curare. — Dissolve  1  part  of  curare  in  100  parts  water,  and 
add  100  parts  of  glycerin.  Of  this  mixture  add  from  5  to  10 
drops  (according  to  the  size  of  the  larva),  or  even  more  for 
large  larvae,  to  a  watch- glassful  of  water.  From  half  to  one 
hour  of  immersion  is  necessary  for  curarisation.  The  larvae 
need  not  be  left  in  the  solution  until  they  become  quite 
motionless;  as  soon  as  their  movements  have  become  slow 
they  may  be  taken  out  and  placed  on  a  slide  with  blotting- 
paper.  If  they  be  replaced  in  water  they  return  to  the 
normal  state  in  eight  or  ten  hours,  and  may  be  re-curarised 
several  times. 

Etherisation. — Three  per  cent,  alcohol  or  3  per  cent,  ether 
may  be  used  in  a  similar  way.  These  reagents  cause  no 


358  CHAPTER    XXVII. 

obstruction  to  the  processes  of  cell-division,  and  are  useful, 
but  their  action  as  anaesthetics  is  inconstant. 

Indifferent  Media. — One  per  cent,  salt  solution,  iodised 
serum,  syrup,  cold  water  (+  1°  C.),  and  warm  water  (35° — 
40°  C.).  The  tail  may  be  excised  from  the  living  animal  and 
studied  for  a  long  time  in  these  media  (PEREMESCHKO,  Arch, 
f.  mik.  Anat.,  xvi,  1879,  p.  437). 

Small  and  transparent  aquatic  organisms,  such  as  larvaa  of 
Diptera,  small  specimens  of  Clepsine  and  Neplielis,  etc.,  may 
be  studied  alive  in  a  reversible  compressorium.  The  vege- 
table kingdom  also  affords  some  good  objects,  for  which  see 
the  botanical  treatises. 

For  the  processes  of  staining  living  cells,  which  are  often 
important  aids  to  study,  see  §  201. 

646.  Study  of  Fresh  and  Lightly  fixed  Cells. — So-called 
"  indifferent "  liquids  must  not  be  believed  to  be  without 
action  on  nuclei.  Iodised  serum,  salt  solution,  serum,  aqueous 
humour,  lymph,  better  deserve  the  name  of  weak  hardening 
agents.  Between  these  and  such  energetic  hardening  agents 
as  Flemming's  mixture  come  such  light  fixing  agents  as 
picric  acid  or  very  dilute  acetic  acid.  These  it  is  whose 
employment  is  indicated  for  the  study  of  fresh  isolated  cells. 

A  typical  example  of  this  kind  of  work  is  as  follows  : — 
Tease  out  a  piece  of  living  tissue  in  a  drop  of  acidulated  solu- 
tion of  methyl  green  (0'75  per  cent,  of  acetic  acid).  This  is 
a  delicate  fixing  agent,  killiDg  cells  instantly  without  change 
of  form.  Complete  the  fixation  by  exposing  the  preparation 
for  a  quarter  of  an  hour  to  vapour  of  osmium,  and  add  a  drop 
of  solution  of  Kipart  and  Petit  and  a  cover. 

Or  you  may  fix  the  preparation,  after  teasing,  with  vapour 
of  osmium  for  half  a  minute  to  two  minutes,  then  add  a  drop 
of  methyl  green,  and  after  five  minutes  wash  out  with  1  per 
cent,  acetic  acid,  and  add  solution  of  Ripart  and  Petit  and 
cover. 

Or  you  may  kill  and  fix  the  cells  by  teasing  in  solution  of 
Ripart  and  Petit  (to  which  you  may  add  a  trace  of  osmic  acid 
if  you  like),  and  afterwards  stain  with  methyl  green. 

I  have  found  Pictet's  chloride  of  manganese  (§  391)  useful 
as  an  examination  medium,  A  little  solution  of  dahlia  may 
be  added  to  it. 


CYTOLOGICAL    METHODS.  359 

HENKING'S  mixture,  which  has  been  given  above  (§  625), 
may  also  be  found  useful. 

Other  fixing  agents,  such  as  picric  acid  or  weak  sublimate 
solution,  may  of  course  be  used,  and  in  some  cases  doubtless 
should  be  preferred.  Other  stains,  too,  such  as  Bismarck 
brown,  may  be  used  as  occasion  dictates ;  and  of  course  other 
examination  media  than  solution  of  Ripart  may  be  employed. 
But,  for  general  purposes,  the  methyl-green-osmium-and- 
Ripart's-medium  method  gives  such  good  results,  and  is  so 
very  convenient,  that  it  may  be  called  a  classical  method  for 
the  study  of  fresh  cells. 

647.  Some  Microchemical  Eeactions. — Methyl  green  is  a  test 
for  chromatin,  in  so  far  as  it  colours  nothing  but  the  chro- 
matin  in  the  nucleus.  It  is,  however,  not  a  perfect  test,  for 
the  intensity  of  the  coloration  it  produces  varies  greatly  in 
different  nuclei,  and  may  in  certain  nuclei  be  extremely  weak, 
or  (apparently)  even  altogether  wanting.  In  these  cases  other 
tests  must  be  applied  in  order  to  establish  with  certainty  the 
presence  or  absence  of  that  element.  The  following  sugges- 
tions are  taken  from  CARNOY,  who  is,  I  believe,  the  only 
writer — on  the  zoological  side,  at  all  events — who  has  insisted 
on  the  necessity  of  applying  microchemical  methods  in  a 
systematic  manner  to  the  study  of  cells. 

Chromatin  is  distinguished  from  albuminoids  by  not  being 
soluble,  as  these  are,  in  water  and  in  weak  mineral  acids, 
such  as  O'l  per  cent,  hydrochloric  acid.  It  is  easily  soluble 
in  concentrated  mineral  acids,  in  alkalies,  even  when  very 
dilute,  and  in  some  alkaline  salts,  such  as  carbonate  of  potash 
and  biphosphate  of  soda.  In  the  presence  of  10  per  cent, 
solution  of  sodium  chloride  it  swells  up  into  a  gelatinous  mass, 
or  even,  as  frequently  happens,  dissolves  entirely  (Bio I.  Cell.f 
pp.  208-9).  It  is  only  partially  digestible  (when  in  situ  in 
the  nucleus)  in  the  usual  laboratory  digestion  fluids. 

The  solvents  of  chromatin  that  are  the  most  useful  in 
practice  are  1  per  cent,  caustic  potash,  fuming  hydrochloric 
acid,  or  cyanide  of  potassium,  or  carbonate  of  potash. 
These  last  generally  give  better  results  than  dilute  alkalies. 
They  may  be  employed  in  solutions  of  40  to  50  per  cent, 
strength.  If  it  be  desired  to  remove  all  the  chromatin  from 
a  nucleus  the  reaction  must  be  prolonged,  sometimes  to  as 


360  CHAPTER    XXV11. 

much  as  two  or  three  days,  especially  if  the  operation  be  con- 
ducted on  a  slide  and  under  a  cover-glass,  which  is  the  safer 
plan. 

It  must  be  remembered  that  these  operations  must  be  per- 
formed on  fresh  cells,  for  hardening  agents  bring  about  very 
considerable  modifications  in  the  nature  of  chromatin,  render- 
ing it  almost  insoluble  in  ammonia,  potash,  or  sodic  phosphate, 
etc.  Hydrochloric  acid,  however,  still  swells  and  dissolves 
it,  though  with  difficulty. 

Partial  digestion  may  render  service  in  the  study  of  the 
chromatic  elements  of  nuclei.  Chromatin  resists  the  action 
of  digestive  fluids  much  longer  than  the  albumins  do  ;  so  that 
a  moderate  digestion  serves  to  free  the  chromosomes  from  any 
caryoplasmic  granulations  that  may  obscure  them,  whilst  at 
the  same  time  it  clears  up  the  cytoplasm. 

Concerning  the  microchemistry  of  the  cell  see  further  last  edition ; 
also  CABNOY  &  LEBRUN,  La  Cellule,  xii,  2,  1897,  p.  94  ;  ZIMMERMANN, 
Die  Morphologic  u.  Physiologie  des  Pflanzlichen  Zellkernes,  Jena,  1896 
(treats  also  of  the  animal  cell) ;  HAECZEE,  Praxis  u.  Theorie  der  Zellen- 
und  Befruchtungslehre,  Jena,  1899. 

648.  Cytological  Fixing  Agents. — It  does  not  follow  that  a 
fixing  agent  that  is  good  for  one  element  of  a  cell  is  also  good 
for  all  others.  That  which  is  good  for  cytoplasm  is  not 
necessarily  good  for  the  nucleus,  and  vice  versa. 

As  regards  the  nucleus,  it  is  a  rule  that  admits  of  no 
exception  that  all  fixatives  must  be  acid  ;  for  if  not  they 
will  not  satisfactorily  preserve  -either  chromatin  or  nucleoli. 

For  instance,  bichromate  of  potash,  if  not  rendered  acid, 
should  be  banished  from  the  study  of  nuclei,  because  it 
causes  chromatin  and  nucleoli  to  swell,  so  that  clear  images  of 
them  are  not  obtained.  (I  do  not  myself  think  that,  as 
regards  chromosomes  at  all  events,  the  images  given  by  bi- 
chromate are  so  unnatural  as  they  are  held  to  be  by  Flemming 
and  the  majority  of  authors.  Chromosomes  during  life  are 
always  in  a  state  that  may  be  fittingly  described  as  swollen 
by  comparison  with  their  state  after  fixation  by  acids. 
During  life,  in  the  equatorial  and  polar  stages  of  division, 
they  are  mostly  compacted  into  plates  or  pectiniform  figures 
in  which  the  separate  elements  are  not  clearly  discernible, 
and  which  are  more  like  the  images  given  by  fixation  with 


CYTOLOGICAL    METHODS.  361 

bichromate  than  those  which  are  given  by  fixation  with  acids. 
The  acids  contract  them  somewhat,  and  so  give  them  sharper 
outlines,  and  thus  render  them  individually  distinguishable. 
The  resulting  image  thus  becomes  clearer,  but  I  do  not 
admit  that  it  is  in  all  cases  more  lifelike.) 

The  fixatives  chiefly  employed  for  nuclei  are  liquid  of 
Flemming  and  liquid  of  Hermann.  For  most  purposes  I  think 
they  are  as  good  as  anything  that  has  hitherto  been  imagined. 
There  is  a  slight  difference  between  them.  Liquid  of  Her- 
mann, owing  to  the  platinum  chloride,  causes  chromatin  to 
shrink  more  than  liquid  of  Flemming  does,  and  thus  often 
gives  clearer  images  of  chromosomes,  especially  of  their 
splitting. 

But  it  is  a  mistake  to  suppose  that  equally  good  images 
cannot  be  obtained  by  means  of  other  reagents.  Some  of  the 
finest  chromosomes  I  have  seen  have  been  fixed  with  Lindsay 
Johnson's  mixture  (§  49),  and  liquid  of  Tellyesniczky  has 
given  me  others  nearly  if  not  quite  as  good. 

Though  I  have  not  found  anything  superior  to  these,  I  do 
not  mean  to  imply  that  there  are  not  others  as  good  or  nearly 
as  good.  Very  likely  there  are ;  for  the  nucleus  is  by  far 
the  easiest  thing  in  the  cell  to  fix  (?'.  e.  so  far  as  chromatin 
and  nucleoli  are  concerned  ;  I  have  left  the  caryoplasm,  or 
whatever  else  there  may  be  in  a  nucleus,  out  of  account,  as 
next  to  nothing  is  known  concerning  it) .  Mixture  of  Gilson- 
Carnoy-Lebrun  gives  very  fair  nuclei  indeed,  and  will  be  found 
highly  useful  where  very  great  penetration  is  required. 

As  regards  the  cytoplasm.  .Cytoplasm  is  made  up  of  two 
elements,  a  fibrillar  network — the  spongioplasm,  reticulum, 
or  mitome ;  and  a  more  or  less  granular  liquid  that  bathes 
it — the  hyaloplasm  or  enchylema.  It  does  not  follow  that 
a  reagent  that  will  fix  one  of  these  will  also  fix  the  other. 
Nor  does  it  follow  that  if  both  are  fixed  you  have  of 
necessity  a  perfect  fixation,  for  that  depends  on  the  object 
in  view. 

If  you  fix  both,  you  will  have  &  full  fixation;  but  in  that 
case  the  granules  of  the  hyaloplasm  (be  they  vital,  or  be  they 
only  "precipitation  forms/'  see  §  27  A),  and  the  secretions 
or  other  inclosures  that  may  be  present  in  it,  may  so  mask 
the  fibrils  of  the  spongioplasm  as  to  interfere  with  the 
observation  of  it.  So  that  if  the  latter  is  the  principal 


V 

362  CHAPTER    XXVII. 

object  of  study,,  a  thin  fixation,  one  in  which  the  spongioplasm 
is  entirely  preserved ,  but  the  hyaloplasm  only  partly,  may  be 
the  better. 

The  spongioplasm  is  the  easier  to  fix  of  the  two,  and  the 
majority  of  acid  fixatives  will  preserve  it  more  or  less ;  for 
instance,  the  osmic  acid,  chromic,  or  picric  mixtures,  or  cor- 
rosive sublimate.  The  best  images  I  have  obtained  are  those 
given  by  liquid  of  Flemming  or  Hermann  in  cells  in  which 
the  action  of  the  reagent  has  been  moderate,  i.  e.  insufficient 
to  thoroughly  fix  the  hyaloplasm  at  the  same  time.  I  have 
also  had  good  results  with  vom  Rath's  picro-osmic  and  picro- 
platinosmic  mixtures,  and  with  acid  sublimate. 

Hyaloplasm  is  not  nearly  so  easy  to  fix,  and  there  are 
only  two  reagents  in  common  use  that  readily  give  a  really 
full  fixation  of  it ;  these  are  osmic  acid  and  bichromate  of 
potash. 

Osmic  acid  acts  as  a  fixative  of  hyaloplasm  in  liquid  of 
Flemming  or  Hermann,  but  only  gives  a  full  fixation  in  the 
outer  layers  of  the  material ;  and  in  these  it  easily  happens 
that  many  or  most  of  the  cells  are  ruined  by  over-fixation 
(see  §§  28,  39). 

This  defect  may  be  to  a  certain  degree  corrected  by  taking 
the  osmic  acid  weaker  than  is  usual.  Thus  by  successively 
reducing  the  proportion  of  this  ingredient  in  liquid  of 
Hermann,*  I  have  found  that  it  can  be  brought  down  to 
one  eighth  of  the  prescribed  amount  without  loss  of  the  dis- 
tinctive characters  of  the  fixation.  But  it  cannot  be  entirely 
omitted  without  the  character  of  the  fixation  changing 
altogether. 

*  NIESSING  (Arch.  f.  mik.  Anat.,  xlvi,  1895,  p.  147)  has  the  following 
two  modifications  oif  Hermann's  mixture  : 

(1)  Platinic  chloride,  10  per  cent,  solution        .         .     25 
Osmic  acid,  2  per  cent.        .         .         .         .         .20 

Glacial  acetic  acid 5 

Distilled  water  . 50 

(2)  The  same  with  saturated  aqueous  solution  of  corrosive  subli- 
mate instead  of  the  water. 

They  are  both  of  them,  in  my  opinion,  as  ill-imagined  as  possible. 
They  contain  some  three  times  as  much  platinic  chloride  as  Hermann's  ; 
and  Hermann's  contains  already  quite  as  much  as  it  can  bear,  and,  I  think, 
much  more  than  is  advisable  :  see  the  proportions  in  the  mixtures  §§  49 
and  53.  RABL  (Anat.  Anz.,  iv,  1889,  p.  21)  employed  it  of  from  y1^  to  ^  per 
cent,  strength,  which  seems  to  me  much  nearer  the  mark. 


OYTOLOGICAL    METHODS.  363 

The  defect  of  want  of  penetration  seems  to  be  incurable 
(see  §§  28,  39,  and  46).  Substitution  of  more  highly  pene- 
trating reagents,  such  as  picric  acid,  for  the  chromic  acid 
or  platinic  chloride,  does  not  help  in  the  least ;  you  only  get 
the  osmic  fixation  outside,  no  whit  deeper  than  before,  and  a 
picro- acetic  fixation,  instead  of  a  chromo-  or  platino-acetic 
one,  in  the  deeper  layers,  fhat  is  all.  For  instance,  vom 
Rath's  picro -platinosmic  mixture,  §  97,  may  often  give  better 
results  in  some  respects  than  liquid  of  Hermann  ;  but  that  is 
not  on  account  of  the  addition  of  the  picric  acid,  it  is  rather 
on  account  of  the  platinic  chloride  being  taken  weaker. 
The  osmic  fixation  is  not  in  the  least  modified  by  the  picric 
acid  in  it. 

In  view,  then,  of  these  defects  of  osmic  mixtures,  it  may 
often  be  advisable,  where  hyaloplasm,  or  its  inclosures,  is  the 
chief  object  of  study,  to  have  recourse  to  bichromate  of  potash. 
The  formula  that  has  given  me  the  finest  fixations  is  that  of 
LINDSAY  JOHNSON,  §  49,  but  it  has  the  drawback  that  there  is 
risk  of  osmication  in  the  outer  layers. 

In  this  respect  liquid  of  Telly esniczky,  §  56,  is  to  be  pre- 
ferred. 

Corrosive  sublimate  gives  a  fairly  full  fixation  ;  but  I 
believe  it  sometimes  produces  serious  artefacts,  HEIDENHAIN'S 
"  Lanthanin  "  being  one  of  them.  I  have,  however,  obtained 
with  liquid  of  Gilson-Carnoy-Lebrun  some  most  excellent 
fixations  of  cytoplasm,  and  I  think  that  the  aqueous  solutions 
of  sublimate  may  frequently  be  used  in  preference  to  liquid 
of  Flemming  on  account  of  the  facilities  they  afford  for  the 
employment  of  certain  stains. 

ALTMAXN'S  fixatives  for  nuclei  see  last  edition,  or  Arch.  Anat.  Entw., 
1892,  p.  223,  and  his  Elementarorganismen,  1890.  His  mixture  for  his 
granula  see  §  48. 

649.  Chromatin  Stains. — For  fresh  tissues  see  §  646. 

For  sections  of  hardened  tissues,  stains  should  be  chosen 
amongst,  those  that  give  a  very  intense  as  well  as  a  very 
sharp  coloration.  Some  years  ago  safranin  and  gentian 
violet,  §  272,  273,  were  the  most  used.  At  the  present 
time  their  place  has  been  taken  by  the  iron  haematoxylin  of 
BENDA  or  HEIDENHAIN. 

To  these  I  think  Kernschwarz  should  be  added. 


364  CHAPTER    XXVII. 

For  some  remarks  of  BATAILLON  and  KOEHLEE  on  the  stain  of  borax- 
methylen-blue  see  Comptes  Eendus,  cxvii,  1893,  p.  521,  or  Journ.  Roy. 
Hie.  8oc.,  1894,  p.  41. 

650.  Plasma  Stains. — I  have  been  unable  to  discover  a 
single  thoroughly  satisfactory  one.  All  of  those  known  to  me 
are  of  an  imperfect  electivity,  in  so  far  as  it  is  difficult  if  not 
impossible  to  limit  their  action  with  the  desired  precision  and 
certitude  to  the  element  that  it  is  desired  to  bring  into  promi- 
nence by  staining.  Almost  all  of  them  colour  too  readily 
the  enchylema  or  hyaloplasm  at  the  same  time  as  the  plas- 
matic  reticulum.  And,  on  the  other  hand,  there  are  many 
important  elements  of  cells  which  cannot  be  got  to  stain 
sufficiently. 

For  Kernschwarz  see  §  365. 

Flemming's  Orange  Method,  §  283,  has  been  much  used. 
I  do  not  recommend  it,  as  it  is  very  capricious  and  unreliable. 
Benda's  Safranin  and  Lightgriin  or  Saureviolett,  §  301,  gives 
sometimes  splendid  results,  but  is  capricious. 

For  Saurefuchsin  and  Orange  G-  see  §§  287,  288. 

Ehrlich-Biondi  mixture  is  a  celebrated  plasma  stain.  It 
is  of  no  use  whatever  for  polar  corpuscles  or  spindle  relics. 
See  §  290. 

The  Osmic  Acid  and  Pyrog allot  Process,  §  361,  gives  a 
very  fair  and  frequently  useful  plasma  stain ;  but  I  do  not 
consider  it  to  be  a  method  of  quite  the  first  class. 

The  Iron-Haematein  Lakes  of  Benda  and  M.  Heidenhain 
give  good  plasma  stains,  according  to  the  degree  of  extrac- 
tion. These  are  the  stains  most  used  for  the  study  of  the 
bodies  known  as  centrosomes,  central  corpuscles,  centrioles, 
polar  corpuscles,  etc.  See  §  255. 

It  is  said  by  Heidenhain,  and  by  other  observers  who 
have  repeated  his  observations,  that  the  stain  is  obtained  in 
a  sharper  form  by  combining  the  hgematein  stain  with  a  fore- 
going stain  with  Bordeaux  R.  He  directs  (Arch.  f.  mik.  Anat., 
xlii,  1894,  p.  665)  that  the  sections  (sublimate  sections  were 
used  by  him)  are  to  be  stained  for  twenty-four  hours  or  more 
in  "a  weak  "  solution  of  Bordeaux,  until  they  have  attained 
such  an  intensity  of  colour  as  that  "  they  would  just  be  fit 
for  microscopic  examination  with  high  powers"  (1.  c.,  p.  440, 
note),  and  that  they  be  then  brought  into  the  ferric  alum. 
After  mordanting  and  staining,  the  hgematem  is  to  be 


CYTOLOGICAL    METHODS.  365 

extracted  in  the  iron  alum  until  the  chromatin  has  become 
entirely  or  almost  entirely  colourless.  Instead  of  Bordeaux, 
"anilin  blue  "  may  be  used  in  the  same  way. 

See  also  Ehrlich's  triacid,  §  291,  and  his  acidophiloiis 
mixture,  §  309 ;  also  Wasserblau,  §  312,  and  other  reagents 
mentioned  in  Chap.  XVI. 

Also  Gold  Chloride,  Apathy's  process,  §  358. 

HERMANN  (Arch.  /.  mik.  Anat.,  xxxvii,  4,  1891,  p.  583)  recommends  a 
modification  of  the  haematoxylin  impregnation  method  of  PAL,  for  which 
see  previous  editions  ;  also  his  paper,  "  Methoden  zum  Studium  des  Archo- 
plasmas  und  der  Centrosomen  tierischer  und  pflanzlicher  Zellen,"  in 
Ergebnisse  der  Anatomie,  Band  ii,  1892  (1893),  p.  23. 

For  HEIDENHAIN'S  Vanadium  hsernatoxylin,  see  COHN  in  Anat.  Hefie, 
1895,  p.  302,  or  Zeit.f.  wiss.  Mile.,  xii,  3,  1896,  p.  359. 

651.  Cell  Granules. — For    the    study    of    the    conspicuous 
"granules/"  undoubtedly  metabolic   products,  occurring  in 
certain  gland-cells  and  blood-  and  lymph- corpuscles,  and  in 
certain  elements  belonging  to  the  group  of  connective  tissues, 
see  the  sections  on  "  Connective  Tissues."    The  most  generally 
employed  stains  are  the  mixtures  of  Ehrlich. 

For  ALTMANN'S  "  Bioblasts  "  see  previous  editions,  also  the  critique  of 
FISCHER,  in  his  Fixirung,  Fdrbung,  und  Ban  des  Protoplasmus,  pp.  108, 
295. 

652.  Nucleoli  are   "  acidophilous "    in   so   far  as,  in   fixed 
material,  they  select  the  acid  dye  or  dyes  from  mixtures  such 
as  the    Ehrlich-Biondi  stain.      With  this  they  stain  mostly 
red,  sometimes  orange.      With  fresh  material   they  do   not 
stain    at    all    with    acid    methyl    green     (distinction    from 
chromatin). 

But  in  fixed  material  treated  with  basic  dyes  (safranin, 
gentian,  etc.)  by  the  regressive  method  they  stain  more 
energetically  than  resting  chromatin,  and  at  least  as  much 
so  as  chromatin  in  the  kinetic  state.  With  iron-haematoxylin 
they  stain  sometimes  full  black,  sometimes  grey  with  a  black 
shell. 

They  can  frequently  be  well  demonstrated  in  unstained 
preparations  examined  in  water,  being  brought  out  by  their 
superior  refractivity. 


CHAPTER  XXVIII. 

TEGUMENTAEY  ORGANS. 

653.  Epithelium. — Both  for  surface  views  and  for  sections 
good  results  are  obtained  by  the  nitrate  of  silver  method,,  the 
methylen  blue  method,  the  perchloride  of  iron  and  pyrogallol 
method  of  the  Hoggans,  §  362,  the  osmic  acid  and  pyrogallol 
process,,  §  361,  and  by  iron-hsematoxylin. 

HEIDENHAIN'S  fixative  for  intestinal  epithelia  may  be  useful 
for  soft  epidermis ;  it  consists  of  a  saturated  solution  of  sali- 
cylic acid  in  one-third  alcohol  (Sitz.  Ber.  Phys.  med.  G-es. 
Wiirzburg,  26th  January,  1899). 

For  the  purpose  of  separating  the  epidermis  from  the 
corium,  LOEWY  (Arch.  f.  mik.  Anat.,  xxxvii,  1891,  p.  159  ; 
Zeit.  f.  wiss.  Mik.,  viii,  2,  1891,  p.  222)  recommends  macerat- 
ing for  twenty-four  to  forty-eight  hours,  at  a  temperature  of 
about  40°  C.,  in  6  per  cent,  pyroligneous  acid.  Acetic  acid 
of  ^  per  cent.  (PHILIPPSON)  is  also  good. 

For  ciliated  epithelium  see  the  methods  of  Englemann 
under  "  Mollusca." 

654.  Intercellular  Bridges  (and  Canals). — On   this    subject 
(which  includes  the  so-called  "  Prickle-cells  ")  see  the  im- 
portant memoirs  of  IDE,  in  La  Cellule,  iv,   2,  1888,  p.  409, 
and  v,  2,  1889,  p.  321;  also  KOLOSSOW,  Arch.  f.  mik.  Anat., 
Hi,    1898,    p.    1;    Zeit.  f.   wiss.  Mik.,  xv,    1,    1898,   p.    92. 
KOLOSSOW  used  an  osmic-acid-tannin  stain,  §  361 ;  I  would 
suggest  that  iron-haematoxylin  ought  to  be  useful. 

See  also  FLEMMING,  Anat.  Hefte,  1  Abth.,  vi,  1895,  p.  1. 

655.  Keratohyalin. — See  UNNA,  Monatsschr.  prakt.  Dermat.,  xx,  1895, 
p.  69 ;  Zeit.f.  wiss.  Mik.,  xiii,  1897,  p.  337. 


TEGUMENTARY    ORGANS.  367 

656.  Plasma-fibrils  of  Epithelium. — KROMAYER'S  process  (Arch, 
f.  mik.  Anat.,  xxxix,  1892,  p.  141 ;  Zeit.  f.  wiss.  Mik.,  ix,  1, 
1892,  p.  84,  and  ix,  3,  1893,  p.  355)  is  as  follows  :— Sections 
are  stained  for  five  minutes  in  a  mixture  of  equal  volumes 
of  anilin  water  (p.  203)  and  concentrated  aqueous  solution 
of  methyl  violet  6  B.  They  are  well  washed  in  water  and 
treated  with  solution  of  iodine  in  iodide  of  potassium  until 
they  become  blue-black  (one  to  thirty  seconds).  They  are 
again  washed  with  water,  dried  with  blotting-paper,  and 
treated  with  a  mixture  of  1  vol.  of  anilin  to  2  vols.  of  xylol 
until  sufficiently  differentiated,  when  they  are  brought  into 
pure  xylol.  Very  thin  sections  will  require  more  xylol  in 
proportion  to  the  anilin,  viz.  1  :  3  or  1  :  4 ;  thicker  ones  may 
require  more  anilin,  viz.  3  :  5  or  3  :  3.  Gentian  or  Kry  stall  - 
violett  will  do  instead  of  methyl  violet,  but  not  quite  so  well. 
For  some  variations  see  Dermatol.  Zeit.,  iv,  1897,  p.  335; 
Zeit.  f.  wiss.  Mik.,  xiv,  3,  1897,  p.  396  ;  further,  EHRMANN 
and  JADASSOHN,  Arch.f.  Dermatol.  u.  Syphilis,  1892,  1,  p.  303 ; 
Zeit.  f.  iviss.  Mik.,  ix,  1893,  p.  356 ;  HERXHEIMER  u.  MULLER, 
Arch.  f.  Dermatol.,  xxxvi,  1896,  p.  93;  Zeit.  f.  wiss.  Mik., 
xiv,  2,  1897,  p.  216  (they  used  Weigert's  Neuroglia  stain)  ; 
SCHUTZ,  ibid.,  pp.  Ill,  218;  and  HERXHEIMER,  Arch.  mik. 
Anat.,  liii,  1899,  p.  510. 

For  the  same  object  UNNA  (Monatssch.  f.  prakt.  Dermatol., 
xix,  1894,  p.  1  and  p.  277,  et  seq. ;  Zeit.f.  wiss.  Mik.,  xii,  1, 
1895,  pp.  61,  63)  has  given  a  whole  series  of  minutely 
detailed  methods,  from  which  the  following  are  some  ex- 
tracts. 

1.  WASSERBLAD-ORCEIN. — Stain    sections  for  ten   minutes 
in  a  neutral  aqueous  1  per  cent,  solution  of  Wasserblau,  rinse 
with  water  and  stain  for  five  or  ten  minutes  in   a   neutral 
alcoholic  1  per  cent,  solution  of  Griibler's  orcein.      Dehydrate, 
clear,  and  mount  in  balsam.      This  method  may  be  varied  as 
follows  : 

(a)  Ten  minutes  in  the  Wasserblau  and  thirty  minutes  or 
more  in  the  orcein. 

(b)  Take  for  the  second  stain  an  acid  solution  of  orcein. 

(c)  Stain  for  only  one  minute  in  the  Wasserblau,  but  for 
thirty  or  more  in  the  neutral  orcein. 

2.  Stain  for  half  an  hour  or  more  in  a  strong  solution  of 
haemalum,  rinse  with  water,  stain  for  half   a  minute   in  a 


368  CHAPTER   XXVIII. 

saturated  aqueous  solution  of  picric  acid,  and  dehydrate  for 
thirty  seconds  in  alcohol  containing  0'5  per  cent,  of   picric 

acid. 

3.   Hsemalum  for  two  hours,  neutral  orcein  as  above  for 

ten  to  twenty  minutes. 

657.  Horn,  Hair,  and  Nails. — The  elements  of    hairs   and 
nails  may  be  isolated  by  prolonged  maceration   in   40   per 
cent,  potash  solution,  or  by  heating  with  concentrated   sul- 
phuric acid.      See  also  VON  NATHUSIUS,  Zool.  Anz.,  xv,  1892, 

p.  395. 

Horny  tissues  stain  well  in  safranin  or  gentian  violet 
(REINKE,  Arch.  f.  mik.  Anat.,  xxx,  1887,  p.  183  ;  ERNST,  ibid., 
xlvii,  1896,  p.  669;  RABL,  ibid.,  xlviii,  1896,  p.  489). 

658.  Skin-nerves  and  Nerve-endings. — Impregnate  with  gold 
chloride.      See  previous  editions,  and  the  methods  given  in 
Chap.  XVIII. 

659.  Tactile  Corpuscles. — FISCHER    (Arch.   f.    mik.    Anat., 
1875,   p.   366)    employed    the    gold   method    of   Lowit — see 
§   351.      RAN  VIE  R    (Traite,    p.    918)    also   recommends    this 
method,  as  well  as  his  two  gold  methods,  §§  352,  353.      See 
RANVIEE,   Traitv,  p.  919;  LANGERHANS,  Arch.f.  mik.  Anat., 
1873,    p.    730;     KULTSCHIZKY,    ibid.,    1884,    p.     358;     and 
SMIRNOW,    Intern.   Monatsschr.   f.   Anat.,   etc.,    x,    1893,    6, 
p.  241  ;   Zeit.  f.  wiss.  Mik.,  x,  2,  1893,  p.  254  (this  observer 
recommends,  beside  the   gold  method  of  Lowit,    the   rapid 
bichromate  of  silver  method  of  Grolgi). 

660.  Corpuscles  of  Herbst  and  Corpuscles  of  Grandry. — DOGIEL 
(Arch.f.  Anat.  .u.  EntwickeL,  1891,  p.  182  ;  Zeit.f.  wiss.  Mik., 
viii,    4,    1892,   p.   520)    prefers   the   methylen  blue   method 
(Chap.   XVII).      Four  per  cent,  solution  of  methylen  blue, 
warmed  to  40°  C.,  is  injected  into  blood-vessels  of  the  heads 
of   ducks  or  geese  ;    pieces  of  skin  are    removed  from  the 
beaks,   sectioned   in  pith,   and   the   sections  brought  on  to 
slides  and  moistened  with  aqueous  or  vitreous  humour  from 
the  animal,  and  left  for  a  few  minutes  exposed  to  the  air  (it 
is  well   to   add  to   the   aqueous   or  vitreous   humour   a  few 
drops  of  y'-g-  per  cent,  methylen  blue  solution).      After  about 
ten  to  thirty  minutes  the  nerve-endings  are  seen  to  be  stained, 


TEGUMENTARY    ORGANS.  369 

and  the  sections  are  then  brought  into  picrate  of  ammonia, 
and  treated  as  described  in  the  chapter  on  "  Methylen  Blue." 
GEBERG  (ibid.,  x,  2,  1893,  p.  244)  has  also  employed  this 
method.  He  has  also  made-  use  of  simple  osmic  acid, 
and  of  the  gold  method  of  ARNSTEIN,  for  which  see  previous 
editions. 

661.  Corpuscles  of  Meissner  and  of  Krause  (Cornea  and  Con- 
junctiva Bulbi  and  Palpebrarum). — DOQIEL  (Arch.f.  mik.Anat., 
xxxvii,    1891,    p.    602,  and   xliv,    i,    1894,    p.    15)  employs 
the  methylen  blue  method  ;  for  details  see  previous  editions. 

See  also  LONGWORTH'S  methods,  Arch.  mik.  f.  Anat.,  1875, 
p.  655. 

662.  Cornea. — There  are  three  chief  methods  for  the  study 
of  the  corneal  tissue — the  methylen  blue  method,  the  silver 
method,  and  the  gold  method. 

For  the  methylen  blue  method  see  Chap.  XVII,  particularly 
§§  330  and  331. 

Negative  images  of  the  corneal  cells  are  easily  obtained  by 
the  dry  silver  method  (KLEIN).  The  conjunctival  epithelium 
should  be  removed  by  brushing  from  a  living  cornea,  and  the 
corneal  surface  well  rubbed  with  a  piece  of  lunar  caustic. 
After  half  an  hour  the  cornea  may  be  detached  and  examined 
in  distilled  water. 

In  order  to  obtain  positive  images  of  the  fixed  cells  the 
simplest  plan  (RANVJER)  is  to  macerate  a  cornea  that  has 
been  prepared  as  above  for  two  or  three  days  in  distilled 
water.  There  takes  place  a  secondary  impregnation,  by 
which  the  cells  are  brought  out  with  admirable  precision. 

The  same  result  may  be  obtained  by  cauterising  the  cornea 
of  a  living  animal  as  above,  but  allowing  it  to  remain  on  the 
living  animal  for  two  or  three  days  before  dissecting  it  out, 
or  by  treating  a  negatively  impregnated  cornea  with  weak 
salt  solution  or  weak  solution  of  hydrochloric  acid  (His). 

But  the  best  positive  images  are  those  furnished  by  gold 
chloride.  RANVIER  prefers  his  lemon- juice  method  to  all 
others  for  this  purpose  (§  353).  It  is  important  that  the 
cornea  should  not  remain  too  long  in  the  gold  solution,  or  the 
nerves  alone  will  be  well  impregnated. 

RAN  VIE  K  also  recommends  this  method  as  the  best  for  the 
study  of  the  nerves. 

24 


370  CHAPTER   XXVIII. 

HOYER'S  method  has  been  given,  §  355. 

See  also  the  methods  of  KOLLETT,  in  Strieker' s  Handb., 
pp.  1102,  1115,  or  previous  editions;  and  TAKTUFERI,  Anat. 
Anz.,  v,  1890,  p.  524 ;  Zeit.f.  wiss.  Mik.,  vii,  3,  1890,  p.  365, 
and  xi,  3,  1894,  p.  346,  or  previous  editions. 

663.  Crystalline. — LOWE  (Arch.  f.  mik.  Anat.,  1878,  p.  557) 
hardens  the  lens  for  a  year  and  a  half  at  least  in  1  per  cent, 
bichromate  of  potash  :  G-EBHAEDT  (Zeit.  f.  wiss.  Mik.,  xiii, 
1896,  p.  306)  for  one  or  two  days  in  4  to  10  per  cent,  forma- 
lin (the  hardened  lens  is  easily  dissociated  with  needles  into 
its  fibres). 

For  Maceration  you  may  use  sulphuric  acid,  §  543. 


CHAPTER    XXIX. 

MUSCLE  AND   TENDON    (NERVE-ENDINGS). 
Striated  Muscle. 

664.  Muscle-cells. — For  the  study  of  these  and  allied  sub- 
jects see,  inter  alia,  BEHRENS,  KOSSEL,  und  SCHIEFFERDECKER, 
Das  Mikroshop,  etc.,  vol.  ii,  pp.  154 — 161 ;   also,  for  the  ap- 
plication of  the   gold  method  to  the  study  of  muscle-cells, 
SCHAFER,  Proc.   Roy.  Soc.,  xlix,  1891,  p.  280;  or  Journ.  Roy. 
Mic.  Soc.,  1891,  p.  683. 

Iron    haematoxylin    gives    very   fine    images    of    striped 
muscle. 

665.  Nerve- endings. — For    the   study   of  nerve-endings    in 
muscle,  both  motor  and  sensory,  the  four  chief  methods  are 
the  methylen  blue  method,  the  gold  method,  the  silver  method, 
and  the  bichromate  of  silver  method  of  Golgi. 

666.  Nerve-endings — the  Methylen  Blue   Method.  —  BIEDER- 
MANN'S  procedure  for  the  muscles  of  Astacus  has  been  indicated 
in  §  326  (see  also  Zeit.f.  wiss.  Hik.,  vi,  1,  1889,  p.  65).    After 
impregnating  as  there  directed  the  carapace  should  be  opened, 
and  the  muscles  exposed  to  the  air  in  a  roomy  moist  chamber 
for  from  two  to  six  hours,  in  order  that  the  stain  may  differ- 
entiate.     The  abdominal  and  caudal  muscles  are  those  which 
give  the  best  results. 

For  Hydrophilus  piceus,  BIEDERMANN  proceeded  by  inject- 
ing 0'5  c.c.  of  methylen  blue  solution  between  the  ultimate 
and  penultimate  abdominal  rings,  in  the  ventral  furrow,  and 
keeping  the  animals  alive  in  water  for  three  to  four  hours. 
After  this  time  the  thorax  should  be  opened  by  two  lateral 
incisions,  and  the  muscles  of  the  first  pair  of  legs  (which  are 


372  CHAPTER    XXIX. 

the  most  suitable)  removed  and  exposed  to  the  air  for  three 
or  four  hours  in  a  moist  chamber,  and  finally  examined  in 
salt  solution. 

G-ERLACH  (Sitzb.  k.  math.-phys.  CL  k.  bayer.  Akad.  Wiss. 
Munchen,  1889,  ii,  p.  125  ;  Zeit.  f.  wiss.  Mik.,  vii,  2,  1890, 
p.  220)  injected  frogs,  either  through  the  abdominal  vein  or 
through  the  aorta,  with  4  to  5  c.c.  of  a  1  :  400  solution  in  1 
per  cent,  salt  solution,  and  examined  pieces  of  muscle  (pre- 
ferably the  head  and  eye  muscles)  in  serum  of  the  animal, 
afterwards  fixing  the  preparations  with  picrate  of  ammonia 
and  mounting  in  glycerin  jelly. 

The  procedure  of  DOGIEL  has  been  given  in  §  326. 

667.  Nerve-endings— the  Gold  Method. — FISCHER   (Arch.  f. 
mik.  Anat.,  1876;  p.  365)  used  the  gold  method  of  LOWIT, 
§  351. 

BTEDERMANN  in  the  paper  quoted  in  the  last  section  re- 
commends for  Axtacus  a  similar  procedure,  the  preliminary 
treatment  with  formic  acid  being  omitted,  and  the  muscles 
being  put  for  a  couple  of  days  into  glycerin  after  reduction 
in  the  acid. 

RANVIER  (Traite,  p.  813)  finds  that,  for  the  study  of  the 
motor  terminations  of  Vertebrates  the  best  method  is  his 
lemon-juice  and  gold-chloride  process  (§  353).  The  delicate 
elements  of  the  arborescence  of  Kiihne  are  better  preserved 
by  this  method  than  by  the  simple  method  of  Lowit. 

668.  Nerve-endings — the   Silver  Method. — RANVIEU  employs 
it  as  follows  (ibid.,  p.  810) :— Portions  of  muscle  (gastrocnemius 
of  frog)  having  been  very  carefully  teased  out  in  fresh  serum, 
are  treated  for  ten  to  twenty  seconds  with  nitrate  of  silver 
solution   of  2  to  3  per  1000,    and    exposed   to  bright   light 
(direct  sunlight  is  best)  in  distilled  water.       As  soon  as  they 
have  become  black  or  brown  they  are  brought  into  1  per  cent, 
acetic  acid,  where  they  remain  until  they  have  swelled  up  to 
their   normal    dimensions.      They   are    then   examined   in   a 
mixture  of  equal  parts  of  glycerin  and  water. 

This  process  gives  negative  images,  the  muscular  substance 
is  stained  brown,  except  in  the  parts  where  it  is  protected  by 
the  nervous  arborescence,  which  itself  remains  unstained. 


MUSCLE    AND    TENDON    (NERVE-ENDINGS).  373 

The  gold  process  gives  positive  images,  the  nervous  structures 
being  stained  dark  violet. 

669.  Nerve- endings— the  Bichromate  of  Silver  Method. — The 
method  of  GOLGI  has  been  successfully  applied  by  RAMON  Y 
CAJAL   to   the    study  of  the   terminations   of  nerves   and   of 
tracheae  in  the  muscles  of  insects,  and  is  doubtless  susceptible 
of  still  wider  applications.      The  process  used  by  him  is  the 
rapid  one.      For  details  see  Zeit.f.  wins.  Mik.,  vii,  3,  1890,  p. 
332,  or  previous  editions. 

670.  Nerve-endings—other  Methods. — See  previous  editions. 


Tendon. 

671.  Corpuscles  of  Golgi   (RANVIEE,  Traite,  p.  929).— Take 
the   tendon   of    the   anterior  and   superior  insertion  of   the 
gemini   muscles   of  the   rabbit.      Free  it  as  far  as  possible 
from    adherent    muscle-fibres.      Treat    it    according    to  the 
formic  acid  and  gold  method  (§  352),  and  after  reduction  of 
the  gold  scrape  the  tendon  with  a  fine  scalpel,  in  order  to 
remove  the  muscle-fibres  that  mask  the  "  musculo-tendinous 
organs/' 

672.  Corpuscles  of  Golgi    (in   the   tendons   of  the  motores 
bulbi  oculi)  (VON  MARCHES  methods,  Archivio  per  le  Scienze 
Hediche,   vol.   v,   No.    15). — The    enucleated   eyes,   together 
with  their  muscles,  were  put  for  not  less  than  three  days 
into   2    per  cent,  bichromate  of  potash.      The  muscles  and 
tendons  were  then  carefully  dissected  out,  stained  with  gold 
chloride    and    osmic    acid    (GoLGi's    method),    and    by    the 
method  of  MANFREDI,  §  355. 

Mount  all  these  preparations  in  glycerin  (balsam  clears 
too  greatly).  The  methods  only  succeed  completely  during 
fine  sunny  weather. 

See  also  RUFFINI  (Atti  R.  Ace.  Lincei  Roma  Rend.  [5],  i,  1892,  p.  442 ; 
Zeit.  f.  wiss.  Mik.,  ix,  2,  1892,  p.  237),  who  recommends  the  method  of 
Fischer. 


374  CHAPTER   XXIX. 

673.  Corpuscles  of  Golgi  (CiACCio,  Mem.  R.  Ace.  8ci.  Bologna 
[4],  t.  x,  1890,  p.  301;  Zeit.  f.  wiss.  Mik.f  vii,  4,  1891, 
p.  507). — For  Amphibia  the  usual  gold  methods  are  not 
satisfactory,  because  the  ground- substance  of  the  tendon 
takes  the  stain  at  the  same  time  as  the  nerve-endings. 
Pieces  of  tendon  should  be  put  into  O'l  per  cent,  hydro- 
chloric acid  or  0*2  per  cent,  acetic  acid  until  quite  trans- 
parent. They  should  then  be  put  for  five  minutes  into  a 
mixture  of  0*1  per  cent,  gold  chloride  and  O'l  per  cent, 
potassium  chloride.  After  that  they  are  put  back  into  the 
acetic  acid,  and  remain  there  for  a  day  in  the  dark,  and  for 
two  or  three  hours  more  in  the  sunlight.  When  they  have 
become  somewhat  violet  they  are  put  for  a  day  into  O'l  per 
cent,  osmic  acid,  and  finally  mounted  in  Price's  glycerin 
acidulated  with  0*5  per  cent,  of  acetic  or  formic  acid. 


Smooth  Muscle. 

674.  Test  for  Smooth  Muscle  (RETTEEEB,  Comptes  Rend.  Soc. 
Biol.,  iv,  1887,  p.  645;  Journ.  Roy.  Mic.  Soc.,  1888,  p.  843). 
— If   a   specimen   of    tissue  be   fixed   in   a   mixture   of   ten 
volumes  of  90  per  cent,  alcohol  and  one  volume  of  formic 
acid,  well  washed,  and  stained  for  twenty-four  to  thirty-six 
hours  with   alum-carmine,   the  cytoplasm  of   smooth  muscle 
will  be  found  to  be  stained  red,  whilst  connective-tissue  cells 
remain  unstained,  and  are  swollen. 

675.  Smooth    Muscle  —  Isolation    of   Fibres.  —  Methods     of 
SCHWALBE,  see  Arch.  f.  mik.  Anat.,  1868,  p.  394,  or  previous 
editions. 

GAGE'S  methods.— See  Journ.  Roy.  Mic.  Soc.,  1887,  p.  327 ; 
and  §§  525,  537,  and  540. 

MOBIUS,  liquid  for  maceration  of  the  muscle  of  Cardium, 
see  above,  §  536. 

BALLOWITZ,  muscle  of  Cephalopoda,  see  Arch.f.  mik.  Anat., 
xxxix,  1892,  p.  291  ;  Zeit.  f.  wiss.  Mik.,  ix,  3,  1893,  p.  344. 

SCHULTZ  (Arch.  Anat.  Phys.,  Phys.  Abth.,  1895-6,  p. 
521)  puts  smooth  muscle  of  Vertebrates  for  twenty-four 
hours  into  10  per  cent,  nitric  acid,  rinses  with  water,  and 


MUSCLE    AND   TENDON    (NEEVE-ENDINGS).  375 

brings  pieces  for  six  to  eight  days  (in  the  dark  at  first)  into 
a  mixture  of  equal  parts  of  -^  per  cent,  osmic  acid  and 
4-  per  cent,  acetic  acid,  teases,  and  mounts  in  glycerin. 

For  smooth  muscle  of  Vermes,  see  APATHY,  Zeit.  f.  wiss. 
Mik.,  x,  1893,  pp.  36,  319. 

676.  Smooth  Muscle,  Specific  Stain  for  (UNNA,  Monatssch.  f. 
prakt.  Dermatol.,  xix,  1894,  p.  533 ;  Zeit.  f.  wiss.  Mik.,  xii, 
2,    1895,    p.    243). — Sections    stained    for    ten    minutes    in 
polychromatic  methylen  blue  solution,  rinsed  in  water,  and 
brought   for   ten   minutes  into   1    per  cent,   solution  of  red 
prussiate   of   potash.       This    fixes    the    colour,    so   that   the 
sections   will    now   bear    differentiating   with    acid    alcohol. 
They  are  treated  accordingly  with  alcohol  acidified  with   1 
per  cent,  of  hydrochloric  acid  for  about  ten  minutes  (until 
the   collagen  ground  comes  out  white).      Absolute  alcohol, 
essence,  balsam. 

In  the  same  place  see  also  another  stain  with  acid  orcein, 
haematein,  Saurefuchsin,  and  picric  acid. 

677.  Iris  (DoaiEL,  Arch.  f.  mik.  Anat.,  1886,  p.  403). — An 
enucleated  eye  is  divided  into  halves,  and  the  anterior  one 
with  the  iris  brought  for  some  days  into  a  mixture  of  two 
parts  one  third  alcohol  and  one   part  0*5   per  cent,  acetic 
acid.      The   iris   can   then   be  isolated,  and   split   from  the 
edge  into  an  anterior  and  posterior  plate,  and  these  stained 
according  to  the  usual  methods. 

See  also  KOGANEI,  Arch.  f.  mik.  Anat.,  1885,  p.  1  ;  Journ. 
Roy.  Mic.  Soc.,  1886,  p.  874. 

Also  CANFIELD,  in  Arch.  f.  mik.  Anat.,  1886,  p.  121  ;  and 
DOSTOIEWSKY,  ibid.,  p.  91. 

678.  Bladder  of  Frog,  Innervation  of  (WOLFF,  Arch.  f.  mik. 
Anat.,    1881,    p.    362). — Impregnation   with    gold   chloride 
injected  into  the  bladder  through  the  anus. 

For  details  see  previous  editions. 

RANVIEB  (Traite,  p.  854)  also  recommends  one  or  the 
other  of  his  two  gold  processes,  the  bladder  being  carefully 
distended  by  injection  of  the  lemon-juice  or  gold  chloride 
and  formic  acid  through  the  cloaca. 


376  CHAPTER  XXIX. 

See  also  the  method  followed  by  BERNHEIM,  Arch.f.  Anat. 
u.Physiol.,  Physiol.  Abth.,  1892,  Supp.,  p.  29;  Zeif.f.  wiss. 
Nik.,  x,  4,  1893,  p.  484 ;  likewise  a  gold  method. 

679,  Stomach  of  Triton  (see  STILLING  and  PFITZNEK,  in  Arch, 
mik.  Anat.,  1886,  p.  396). 


CHAPTER  XXX. 

NEUROLOGICAL   METHODS— INTRODUCTION    AND    SECTION 
METHODS. 

680.  Introduction. — Histological  research  into  the  structure 
of  the  nervous  system  pursues  two  ends.  Either  it  is  desired 
to  elucidate  the  minute  structure  of  the  nervous  elements  or 
neurons  (neurites — FISH),  that  is  to  say,  the  internal  organisa- 
tion of  nerve  cells  and  nerve  fibres  :  the  processes  employed 
to  this  end  forming  a  group  of  cytological  methods.  Or  it  is 
desired  to  study  the  form  of  nerve  cells,  the  exact  distribution 
of  the  divers  groups  of  nerve  cells  in  the  grey  matter,  the 
connections  that  are  formed  by  means  of  nerve  fibres  between 
these  groups  of  nerve  cells  or  "  nuclei/'  and  to  follow  out  the 
intricate  course  of  the  tracts  of  fibres  that  enter  into  the 
constitution  of  the  white  matter  of  the  cerebro-spinal  axis. 
The  processes  employed  in  all  these  researches  form  a  group 
of  the  anatomical  methods  of  neurology.  It  is  more  espe- 
cially in  this  group  that  we  find  highly  special  methods  of 
selective  coloration.  This  group  may  be  divided  as  follows  : . 

A.  Nerve  Fibres. 

(a)  Myelin  stains  ;  comprising  the  methods  of  WEIGERT, 
and  similar  methods. 

(6)  Axis-cylinder  stains,  and  axis-cylinder  and  my  el  in 
stains. 

B.  Nerve  Cells. 

(c)  Axis-cylinder-and- protoplasm  stains,  comprising  the 
methylen  blue  method  and  some  rather  old-fashioned  general 
stains. 


378  CHAPTER   XXX. 

(d)  Axis-cylinder-and-protoplasm  impregnations,  consist- 
ing chiefly  of  the  methods  of  GOLGI  (the  sublimate  method 
and  the  three  bichromate  of  silver  methods),  and  certain  gold 
methods. 


A  large  proportion  of  the  methods  used  in  the  study  of 
nerve-tissue  in  peripheral  organs  have  already  been  exten- 
sively treated  of  in  the  chapters  on  "  Methylen  Blue/'  on 
"  Impregnation  Methods/'  on  "  Tegumentary  Organs/'  and 
on  "  Muscle  and  Tendon/'  The  reader  should  therefore 
bear  in  mind  that  a  considerable  part  of  the  subject  properly 
comprehended  under  the  term  "  Neurological  Methods "  is 
contained  in  those  chapters,  which  should  be  referred  to  in 
order  to  complete  the  account  given  in  the  following  pages. 

The  remainder  of  this  chapter  will  be  devoted  to  the 
special  section  methods  employed  for  the  central  nervous 
system,  and  to  the  Cytological  Methods  of  Neurology. 
Group  A  of  the  Anatomical  Methods  will  be  given  in  Chap. 
XXXI,  and  Group  B  in  Chap.  XXXII. 

For  more  minute  details  concerning  the  dissection  and  hardening  of  the 
voluminous  encephala  of  Man  and  the  larger  Vertebrates  than  can  be  given 
here  see  MERCIEE,  Les  Coupes  du  Systeme  Nerveux  Central  (1894,  Paris, 
Rueff) ;  DEJEEINE,  Anatomie  des  Centres  Nerveux,  Paris,  1895  ;  BEVAN 
LEWIS,  The  Human  Brain  ;  Histological  and  Coarse  Methods  of  Research, 
London,  Churchill ;  and  OBEESTEINEB,  Anleitung  beim  Studium  des  Baues 
d.  nervosen  Centralorgane  im  gesunden  u.  kranken  Zustande,  Leipzig, 
Toeplitz. 


SECTION  METHODS. 

681.  Fixation  by  Injection. — Fixation,  in  the  proper  sense 
of  the  word,  is,  of  course,  out  of  the  question  in  the  case  of 
the  human  subject.  But  in  the  case  of  the  lower  animals  it 
is  possible  to  introduce  fixing  liquids  into  the  living  nerve 
centres  by  means  of  injection,  thus  ensuring  a  much  more 
rapid  penetration  of  the  reagents  than  can  be  obtained  by 
simple  immersion.  This  method  was,  I  believe,  first  sug- 
gested by  GOLGI  (Arch.  Ital.  de  Biologie,  t.  vii,  p.  30).  He 
injected  2'5  per  cent,  solution  of  bichromate  of  potash  through 


NEUROLOGICAL    METHODS.  379 

the  carotid  if  he  wished  to  limit  the  hardening  to  the  en- 
cephalon,  or  through  the  aorta  if  he  desired  to  fix  the  spinal 
cord. 

DE  QUERVAIN  (Vircliow's  Archiv,  cxxxiii,  1893,  p.  489 ; 
Zeit.  f.  u'iss.  MiJc.,  x,  4,  1893,  p.  507)  proceeds  in  a  similar 
manner,  injecting  solution  of  Miiller  warmed  to  body  heat. 
For  dogs  300  to  400  c.c.  are  required,  for  cats  one  third  to 
one  half  that  quantity.  After  injection  the  whole  organ  is 
put  into  solution  of  Miiller  for  some  weeks. 

(Further  details  in  last  edition.) 

MANN  (Zeit.  f.  wiss.  Mik.,  xi,  4,  1894,  p.  482)  injects 
through  the  aorta.  Before  throwing  in  the  fixing  liquid,  he 
injects  for  about  twenty  seconds  physiological  salt  solution 
warmed  to  39°  C.  This  washes  out  the  capillaries,  and 
prevents  the  blood  from  coagulating  there.  The  fixing  solu- 
tion employed  by  him  consists  of  saturated  solution  of  corro- 
sive sublimate,  warmed  to  39°  C.  After  five  minutes  of  injec- 
tion the  brain  ought  to  be  fixed  throughout.  It  is  removed 
and  put  for  twelve  hours  into  the  same  sublimate  solution, 
after  which  it  is  either  put  for  permanent  preservation  into  O'l 
per  cent,  solution  of  sublimate,  or  is  at  once  passed  through 
alcohol  for  imbedding  in  paraffin . 

See  also  §  754. 

STRONG  (New  York  Acad.  of  Sci.,  January  13th,  1896; 
Anat.  Anz.t  xi,  21,  1896,  p.  655)  advises  injecting  formalin 
diluted  with  an  equal  volume  of  water,  or  (for  the  GOLGI 
method)  with  an  equal  volume  of  10  per  cent,  solution  of 
potassium  bichromate  ;  which  seems  to  me  very  heroic  (see 
§  104). 

Hardening. 

682.  Hardening  by  the  Freezing  Method.— The  ether  freezing 
method  is  to  be  preferred.  The  sections  should  be  floated  on  to  water, 
treated  for  a  minute  on  the  slide  with  0*25  per  cent,  osmic  acid  solution, 
and  stained  or  otherwise  treated  as  desired. 

For  a  detailed  description  of  these  manipulations  see  BEVAN  LEWIS'S  The 
Human  Brain. 

GOODALL'S  Rapid  Method  for  preparing  Spinal  Cord  (Brit.  Med.  Journ., 
May,  1893,  p.  947  ;  Journ.  Roy.  Mic.  Soc.,  1893,  p.  405).— Cut  sections  of 
fresh  tissue  with  a  freezing  microtome  ;  float  them  on  to  water,  and  as  soon 
as  possible  drain  them  and  float  them  on  to  pjridin.  After  a  quarter  of  an 


380  CHAPTER   XXX. 

hour  wash  in  water ;  stain  with  0'25  per  cent,  aqueous  solution  of  anilin  blue- 
black,  followed  by  picro-carmine  ;  dehydrate  and  clear  in  pyridin  ;  mount 
in  balsam  thinned  with  pyridin.  See  also  §  103. 


683.  Generalities  on  Hardening  by  Reagents. — If  large  pieces 
of  nerve-tissue  are  to  be  hardened,,  it  is  necessary  to  take 
special  precautions  in  order  to  prevent  them  from  becoming 
deformed  by  their  own  weight  during  the  process.  Spinal 
cord  or  small  specimens  of  any  region  of  the  encephalon  may 
be  cut  into  slices  of  a  few  millimetres'  thickness,  laid  out  on 
cotton  wool,  and  brought  on  the  wool  into  a  vessel  in  which 
they  may  have  the  hardening  liquid  poured  over  them.  The 
wool  performs  two  functions  :  it  forms  an  elastic  cushion  on 
which  the  preparations  may  lie  without  being  distorted  by 
their  own  weight ;  and  it  allows  the  reagent  to  penetrate  by 
the  lower  surfaces  of  the  preparations  as  well  as  by  their  ex- 
posed surfaces.  A  further  precaution,  which  is  useful,  is  to 
hang  up  the  preparations,  lying  on  or  in  the  cotton  wool,  in  a 
glass  cylinder  or  other  tall  vessel ;  by  hanging  them  near  the 
top  of  the  liquid  the  processes  of  diffusion  and  the  penetra- 
tion of  the  reagent  are  greatly  facilitated. 

If  the  preparations  are  placed  on  the  bottom  of  the  vessel, 
they  should  never  be  placed  one  on  another. 

If  it  be  desired  to  harden  voluminous  organs  without 
dividing  them  into  portions,  they  should  at  least  be  incised 
as  deeply  as  possible  in  the  less  important  regions.  It  is 
perhaps  better  in  general  not  to  remove  the  membranes  at 
first  (except  the  dura  mater),  as  they  serve  to  give  support 
to  the  tissues.  The  pia  mater  and  arachnoid  may  be  removed 
partially  or  entirely  later  on,  when  the  hardening  has  already 
made  some  progress. 

The  spinal  cord,  the  medulla  oblongata,  and  the  pons  Varolii 
may  be  hardened  in  toto.  The  dura  mater  should  be  removed 
at  once,  and  the  preparation  hung  up  in  a  cylinder-glass 
with  a  weight  attached  to  its  lower  end.  The  weight  has  the 
double  function  of  preventing  any  part  of  the  preparation 
from  floating  above  the  level  of  the  hardening  liquid  (a  thing 
that  easily  happens  where  somewhat  dense  liquids,  such  as 
Miiller's  solution,  are  used),  and  of  preventing  the  torsions  of 
the  tissues  that  may  otherwise  be  brought  about  by  the  elastic 
fibres  of  the  pia  mater  and  arachnoid. 


NEUROLOGICAL  METHODS.  381 

The  cerebrum  should  be  very  delicately  laid  out  on  a  layer 
of  cotton  wool,  or,  if  possible,  hung  up  in  it.  Plugs  of  the 
wool  should  be  put  into  the  fissure  of  Sylvius,  and  as  far  as 
possible  between  the  convolutions.  Unless  there  are  special 
reasons  to  the  contrary,  the  brain  should  be  divided  into  two 
symmetrical  halves  by  a  sagittal  cut  passing  through  the 
median  plane  of  the  corpus  callosum.  BETZ  recommends  that 
after  a  few  hours  in  the  hardening  liquid  the  pia  mater  should 
be  removed  wherever  it  is  accessible,  and  the  choroid  plexuses 
also.  I  have  found  this  by  no  means  easy,  and  think  it  is 
an  operation  that  can  only  be  recommended  for  experienced 
hands. 

The  cerebellum  should  be  treated  after  the  same  manner. 

The  temperature  at  which  the  preparations  are  kept  in  the 
hardening  solution  is  an  important  point.  The  hardening 
action  of  most  solutions  is  greatly  enhanced  by  heat.  Thus 
WEIGERT  (Centralb.  f.  d.  med.  Wiss.,  1882,  p.  819;  Zeit.  f. 
wiss.  Mik.j  1884,  p.  388)  finds  that  at  a  temperature  of  from 
30°  to  40°  C.  preparations  may  be  sufficiently  hardened  in 
solution  of  Miiller  in  eight  or  ten  days,  and  in  solution  of 
Erlicki  in  four  days  ;  whilst  at  the  normal  temperature  two  or 
three  times  as  long  would  be  required. 

But  it  is  not  certain  that  this  rapid  hardening  always  gives 
the  best  definite  results.  SAHLI,  who  has  made  a  detailed 
study  of  the  hardening  action  of  chrome  salts,  is  of  opinion 
that  it  does  not,  and  thinks  it  ought  for  this  reason  to  be 
abandoned  (see  Zeit.  f.  wiss.  Mik.,  1885,  p.  3). 

On  the  other  hand,  the  slowness  of  the  action  of  chromic 
salts  at  the  normal  temperature  is  such  that  decomposition 
may  easily  be  set  up  in  the  tissues  before  the  hardening  and 
preserving  fluid  has  had  time  to  do  its  work.  For  this  reason 
voluminous  preparations  that  are  to  be  hardened  in  the  slow 
way  should  be  put  away  in  a  very  cool  place — best  of  all  in 
an  ice-safe. 

See  also  the  paper  of  PFISTEB  on  the  preliminary  hardening  of  Central 
Nervous  System  in  situ,  in  Neurol.  Centralb.,  xvii,  1898,  p.  643  (Zeit. 
f.  wiss.  Mik.,  xv,  4,  1899,  p.  494). 

684.  The  Reagents  to  be  employed. — The  hardening  agents 
most  used  are  the  chromic  salts.  Chromic  acid  was  much 
used  at  one  time,  but  most  workers  now  agree  that  its  action,. 


382  CHAPTER   XXX. 

though  much  more  rapid  than  that  of  the  salts,  is  much  more 
uneven,  and  frequently  causes  a  disastrous  friability  of  the 
tissues.  Osmic  acid  can  hardly  be  used  for  objects  of  more 
than  a  cubic  centimetre  in  size  at  most. 

It  has  already  been  noted  that  the  liquid  of  Erlicki  has  a 
more  rapid  action  than  the  other  solutions  of  chromic  salts. 
SAHLJ,  however  (loc.  cit.  last  §),  after  having  studied  the 
action  of  the  usual  solutions,  concludes  that  the  best  harden- 
ing agent  for  fresh  tissues  is  pure  bichromate  of  potash,  in 
3  or  4  per  cent,  solution,  the  hardening  being  done  in  a  cold 
place.  He  rejects  the  liquid  of  Erlicki  on  account  of  the 
precipitates  it  so  frequently  gives  rise  to  (see  §  58). 

OBEBSTEINER  is  of  the  same  opinion,  recommending  pure 
bichromate  for  general  hardening  purposes;  whilst  for  the 
study  of  the  most  delicate  structural  relations  he  recommends 
fixing  in  Fol's  modification  of  Flemming's  liquid  (§  46)  for 
twenty-four  hours,  followed  by  washing  with  water  and 
hardening  in  80  per  cent,  alcohol. 

In  view  of  the  slowness  of  penetration  of  chromic  salts,  it  is 
often  advisable  to  treat  preparations  for  twenty-four  hours  or 
more  with  alcohol  of  80  to  90  per  cent  before  putting  them 
into  the  hardening  liquid,  in  order  to  avoid  maceration  of  the 
deeper  layers  of  tissue. 

BUECHABDT  (La  Cellule,  xii,  2, 1897,  p.  337)  says  that  "  according  to  the 
unanimous  judgment  of  all  investigators  the  bichromates  of  potash  and 
ammonia  should  not  be  employed  for  the  cytological  study  of  nerve  cells." 
I  have  not  noticed  any  such  consensus  of  opinion  of  authors. 

Several  observers  have  lately  been  using  acetic  alcohol. 
So  TIMOPEEW,  Intern.  Monatttschr.,  An  at.  u.  Phys.,  xv,  18&8, 
p.  259  ;  Zeit.f.  wiss.  Mik.,  xvi,  1,  1899,  p.  99  (CAKNOY'S  second 
formula,  §  83). 

OHLMACHER  recommends  his  sublimate  mixture,  §  84. 

For  formaldehyde  see  §§  104,  692,  and  752. 

Chromic  acid  is  not  much  used  alone  (see  §  40).  It  forms  part  of  some 
of  the  mixtures  mentioned  below.  A  very  little  chromic  acid  (say  one  to 
two  drops  of  1  per  cent,  solution  for  each  ounce)  added  to  bichromate  solu- 
tion will  do  no  harm,  and  will  quicken  the  hardening. 

Nitric  acid  has  been,  and  still  is,  employed  in  strengths  of  10  to  12  per 
cent.,  and  gives  particularly  tough  preparations. 

Neutral  acetate  of  lead  in.  10  per  cent,  solution  affords  an  excellent  pre- 
servation of  ganglion  cells,  according  to  ANNA  KOTLAEEWSKI  (see  Zeit.  /. 
wiss.  Mile.,  iv,  3,  1887,  p.  387). 


NEUROLOGICAL    METHODS.  333 

TBZEBINSKI  (Virchow's  Arch.,  1887,  p.  1 ;  ZeiLf.  wise.  Mil-.,  iv,  4,  1887, 
p.  497)  finds  that,  as  regards  the  faithful  preservation  of  ganglion  cells  (of 
the  spinal  cord  of  the  rabbit  and  dog),  the  best  results  are  obtained  by 
hardening  for  eight  days  in  7  per  cent,  solution  of  corrosive  sublimate, 
followed  by  hardening  in  alcohol  containing  0'5  per  cent,  of  iodine. 

DIOMIDOFF  (ibid.,  p.  499)  also  obtained  very  excellent  results  by  harden- 
ing small  pieces  of  brain  (as  suggested  by  GAULE,  OGATA,  and  BECHTEEEFF) 
for  from  five  to  nine  days  (not  more  in  any  case)  in  7  per  cent,  sublimate 
solution,  followed  by  alcohol.  (This  process  produces  artificial  "  pigment 
spots,"  similar  to  those  produced  by  solution  of  Erlicki ;  they  may  be  dis- 
solved out  by  prolonged  treatment  with  warm  water,  or  in  five  minutes  by 
strong  solution  of  LUGOL.)  The  tissues  are  of  a  good  consistence  for 
cutting. 

Chloride  of  zinc  has  been  recommended  for  some  purposes  (see  §§  691, 
692). 

Two  recent  observers,  FISH  (The  Wilder  Quarter- Century  Book,  1893, 
p.  335)  and  DONALDSON  (Journ.  ofMorphol.,  ix,  1894,  p.  123  ;  Journ.  Roy. 
Mic.  Soc.,  1894,  p.  642),  have  made  numerous  determinations  of  weight  and 
volume,  with  the  object  of  ascertaining  what  changes  are  produced  by 
reagents  in  encephala  of  sheep.  They  have  found  that  bichromate  of 
potash  produces  a  slight  increase  both  of  weight  and  volume,  whereas  all 
the  other  reagents  tried  produce  a  diminution  of  both  these  factors. 

FLATAU  (Anat.  Anz.,  xiii,  1887,  p.  323)  finds  that  brain  augments  in 
weight  slightly  in  10  per  cent,  forinol  solution  (spinal  cord  somewhat 
more) ;  whilst  in  1  per  cent,  solution  it  may  increase  as  much  as  24  per 
cent. 

685.  Strengths  of  the  Reagents. — All  hardening  reagents 
(except  osmic  acid)  should  at  first  be  taken  as  weak  as  is 
consistent  with  the  preservation  of  the  tissue,  and  be  changed 
by  degrees  for  stronger. 

Osmic  acid  may  be  taken  of  1  per  cent,  strength,  and  will 
harden  small  pieces  of  tissue  sufficiently  in  five  to  ten  days 
(EXNEB). 

Bichromate  of  potash  should  be  taken  at  first  of  not  more 
than  2  per  cent,  strength ;  this  is  then  gradually  raised  to 
3  or  4  per  cent,  for  the  cord  and  cerebrum,  and  as  much  as 
5  per  cent,  for  the  cerebellum.  OBERSTEINER  begins  with 
1  per  cent.,  and  proceeds  gradually  during  six  to  eight 
weeks  to  2  or  3  per  cent.  (This  is  at  the  normal  tempera- 
ture ;  at  a  temperature  of  35°  to  45°  C.  the  hardening  can 
be  got  through  in  one  or  two  weeks.) 

Bichromate  of  ammonia  should  be  taken  of  half  the 
strength  recommended  for  bichromate  of  potash,  or  even 
weaker  at  first ;  it  may  be  raised  to  as  much  as  5  per  cent, 
for  cerebellum  towards  the  end  of  the  hardening. 


384  CHAPTER    XXX. 

686.  BETZ'S  Methods  (Arch.  f.  mik.  Anat.,  1873,  p.  101).—  - 
Brain   and  spinal  cord  are  first  subjected  to  a  preliminary 
hardening,  for   some  days   or  weeks,  in  70  to  80  per  cent, 
alcohol  containing  enough   iodine   to  give  it  a  light  brown 
coloration.       After-  this    they    are    definitely    hardened    in 
bichromate  of  potash  of  3  per  cent,  for  spinal  cord,  medulla 
oblongata,  and  pons,  5  per  cent,  for  cerebellum,  and  4  per 
cent,  for  cerebrum. 

For  details  see  previous  editions. 

687.  Cerebrum  (BEVAN  LEWIS,  The  Human  Brain,  p.  102). 
— Methylated    spirit,    twenty-four    hours    in    a   cool    place. 
Miiller's  solution,  three  days  in  a  cool  place.      Then  change 
the  liquid ;   and  after  three  days  more  change  it  again,  or, 
preferably,   substitute   a   2    per   cent,  solution   of  potassium 
bichromate.      At  the  end  of  the  second  week  a  solution  of 
double  the  strength  may  be  added ;   and  if  at  the  termina- 
tion of  the  third  week  the  mass  is  still  pliable,  and  of  the 
consistence  of  ordinary  rubber,  it  is  as  yet  unfit  for  section 
cutting,  and  the  reagent  should  be  replaced  by  a  solution  of 
chromic  acid. 

688.  Brain  (HAMILTON,  Journ.  of  Anat.  and  PhysioL,  1878, 
p.    254). — Slices    of   fresh  brain   about  one   inch  thick   are 
placed  flat  in  a  large  vessel  padded  with  cotton ;   do  not  put 
them  one  above  the  other.      Cover  them  with  the  following 
fluid  (after  cooling)  : 

Miiller's  fluid  .  .          «;          .          3  parts. 

Methylated  spirit    .  .  .        .          .  1  part. 

Put  the  preparations  away  in  an  ice-safe.  Turn  the 
segments  over  next  day.  Change  the  solution  in  a  fortnight 
or  three  weeks  ;  or  if  on  examining  a  section  of  one  of  the 
pieces  it  is  found  that  the  hardening  reagent  has  penetrated 
to  the  interior,  they  may  be  at  once  removed  to  the 
following  mixture  : 

Bichromate  of  ammonia     .  .  1  grm. 

Water    ......      400  c.c., 

in  which  they  remain  for  one  week.  Then  change  the 
solution  to  one  of  1  per  cent,  for  one  week  ;  and  let  this  be 
followed  by  a  solution  of  2  per  cent,  for  another  week,  or 
longer  if  required.  The  pieces  will  now  be  sufficiently  hard 


NEUROLOGICAL   METHODS.  385 

for  cutting  ;  they  may  be  kept  permanently  in  solution  of 
chloral  hydrate,  twelve  grains  to  the  ounce. 

689.  Entire  Encephalon    (DEECKE,   Journ.   Roy.   Mic.   Soc., 
1883,  p.  449). — Bichromate  of  ammonia  in  £  to  1  per  cent, 
solution,  according  to   the   consistence  of  the  brain.      If   it 
happens  to  be  soft  he  adds  say  %  to  T^  per  cent,  of  chromic 
acid  to  the  solution,  and  always  ^  to  ^  of  the  whole  volume 
of  alcohol. 

Further  details  in  previous  editions. 

690.  Encephalon  (M.  DUVAL,  ROBIN'S  Journal  de  I'Anatomie, 
1876,   p.   497).— First    Method.— Place   the  fresh  tissues  in 
solution   of  bichromate  of   potash    25,  water   1000 ;   change 
the  liquid  after  the  first  twenty-four  hours,  and  again  after 
three  or  four  days.      After   two  or  three   weeks   place   the 
preparations    in    chromic   acid   of   3   per    1000,    change    the 
liquid   every  day  for  the   first  week,  and  after  that  every 
eight    days    until   the   middle   of   the   second   month,   after 
which   time  it  is  no  longer   needful   to   change   the   liquid. 
The  preparations  must  remain  at  least  two  months  in  the 
chromic  acid ;   the  longer  they  remain  in  it  the  better.      A 
few  fragments  of  camphor  should  be  added  to  the  liquid  in 
order  to  prevent  the  growth  of  mould. 

691.  Encephalon  (Fisn,  The  Wilder  Quarter- Century  Boole, 
1893,  p.  393). 

Water 400  c.c. 

95  per  cent,  alcohol          .  .  .          400    „ 

Glycerin 250    „ 

Zinc  chloride  ....  20  grms. 

Sodium  chloride      .  .  .  .  20      ,, 

Immerse  in  this,  filling  the  cavities  of  the  brain  with  it,  and 
if  practicable  also  injecting  the  blood-vessels  with  it,  for 
about  three  days,  then  transfer  for  a  week  or  more  to  a 
mixture  of  equal  parts  of  the  fluid  and  70  per  cent,  alcohol, 
and  finally  store  in  90  per  cent,  alcohol. 

692.  Formaldehyde. — For  formaldehyde   in   general,   see   § 
104. 

WEIGERT    (Beitr.   zur   Kenntn.   d.   normalen   menscklichen 

25 


386  CHAPTER    XXX. 

Neuroglia,  1895,  quoted  from  Neuyol.  Centralb.,  1895,  p. 
1146)  puts  portions  of  material  of  not  more  than  half  a 
centimetre  in  thickness  for  four  days  into  a  "  4  per  cent, 
solution  of  formol  "  (by  which  is  presumably  meant  com- 
mercial formol  diluted  with  9  volumes  of  water). 

MARCUS  (quoted  from  FISH,  see  below ;  see  also  Zeit.  f. 
wiss.  Mik.j  xiii,  1896,  p.  241)  recommends  hardening  the 
spinal  cord  for  two  or  four  weeks  in  a  ^  per  cent,  solution 
of  formalin,  then  small  pieces  one  half-centimetre  thick  are 
cut  out  and  placed  in  Mulleins  fluid  for  a  week  in  an  oven 
at  37°  C. 

VAN  GIUSON  (An at.  Anz.,  x,  1895,  p.  494)  states  that  he 
obtained  good  results  by  using  "  solutions  of  formalin  of  4, 
6,  and  10  per  cent.,"  followed  by  95  per  cent,  alcohol. 
Myelin  was  found  to  be  well  preserved  and  to  give  the 
characteristic  blue  reaction  with  Weigert's  hsematoxylin  (the 
1885  method),  just  as  if  a  chrome  salt  were  present. 

LACHI  (cf.  Zeit.  f.  wiss.  Mik.,  xii,  1895,  p.  32)  states  that 
he  has  had  good  results  with  <(  20  per  cent,  solutions  of 
formol." 

FISH  (Proc.  Amer.  Mic.  Soc.,  xvii,  1895,  p.  319)  recom- 
mends— 

Water ,  .     2000  c.c. 

Commercial  formalin  .  .  .         50    „ 

Sodium  chloride          .  .  .  .100  grms. 

Zinc  chloride    .  .  .  .  15      „ 

Brains  should  be  left  in  this  mixture  for  a  week  or  ten  days 
or  more,  then  transferred  to  a  2'5  per  cent,  solution  of 
formalin  (water  2000  c.c.,  formalin  50  c.c.),  in  which  they 
may  remain  indefinitely  if  the  jar  be  kept  tightly  covered. 

PARKEK  and  FLOYD  (Anat.  Anzeiger,  Bd.  xi,  1895,  No.  5, 
p.  156)  find  that  a  f4  2  per  cent,  solution  of  formol,"  by 
which  is  meant  a  mixture  of  two  volumes  of  formol  with  9§ 
of  water,  will  harden  a  sheep's  brain  in  a  week  or  ten  days 
in  a  satisfactory  manner  as  regards  consistency,  but  with  a 
marked  increase  of  volume,  which  may  amount  to  as  much 
as  40  per  cent. !  To  obviate  this  they  advise  a  mixture  of — 
Alcohol  95  per  cent.  .  .  .6  volumes, 

Formol  2  per  cent,  (the  above  mixture)  4        „ 
which  has  the  same  excellent  and  rapid  hardening  qualities 
and   gives    only  a  .J^ardly  perceptible   increase   of    volume. 


NEUROLOGICAL   METHODS.  387 

Brains  may  be  kept  for  months  in  the  mixture  (ibid.,  1896, 
p.  568). 

GEEOTA  (Zeit.  f.  wiss.  Mik.,  xiii,  1896,  p.  314)  puts  human 
brains  into  a  5  to  10  per  cent,  solution  of  formol,  and  after 
twenty- four  hours  removes  the  pia  and  changes  the  liquid ; 
this  is  also  further  done  every  five  to  seven  days,  and  in  one 
or  two  weeks  the  hardening  is  complete.  In  the  case  of 
foetal  brains  of  Canix,  Fdis,  and  Homo,  he  first  injects  the 
vascular  system  with  a  10  to  15  per  cent,  solution  of  formol 
in  85  per  cent,  alcohol,  and  then  brings  the  heads  into  the 
5  to  10  per  cent,  watery  solution  ;  after  one  or  two  days  he 
removes  the  brains  from  the  skulls  and  puts  them  back  for 
fifteen  to  twenty  days  into  the  formol. 

MABINA  (Riv.  Pat.  Nerv.  Ment.,  Firenze,  ii,  1897,  p.  20 ;  Neur.  Centralb., 
xvi,  1897,  p.  166 ;  Zeit .  f.  wiss.  Mik.,  xiv,  p.  231)  fixes  portions  of  central 
nervous  system  for  four  to  eight  days  in  a  freshly  prepared  mixture  of  100  c.c. 
90  per  cent,  alcohol,  5  c.c.  formol,  and  O'l  gnu.  chromic  acid,  changing  the 
mixture  daily. 

ORTH  (Berl.  klin.  Wochenschr.,  1896,  No.  13;  Zeit.  f.  wiss. 
Mik.,  xiii,  3,  1896,  p.  316)  recommends  under  the  title  of 
Forwol-Muller  (or,  abbreviated,  F.  M.),  a  mixture  of  one  part 
of  formol  to  ten  of  liquid  of  Miiller  (§  57).  It  must  be 
freshly  prepared,  and  changed  every  few  days.  Small  pieces 
of  tissue  may  be  sufficiently  hardened  in  a  few  hours  in  a  stove. 

KRADSS  (Trans.  Amer.  Mic.  Soc.,  xvii,  1896,  p.  315;  Zeit. 
f.  wiss.  Mik.,  xiii,  1896,  p.  493)  finds  that  formalin  in  different 
strengths  hardens  well,  but  causes  a  notable  shrinkage  of 
neuroglia,  and  prefers  liquid  of  Miiller. 

See  also  SIEMKRLING,  NeuroL  Centralb.,  xviii,  1899,  p.  472  ; 
Zeit.  f.  wiss.  Mik.,  xvi,  4,  1900,  p.  470  (10  per  cent,  formol 
gives  a  good  consistency ;  but  if  it  is  allowed  to  act  long, 
stains  will  be  bad  unless  the  material  is  after-hardened  for 
some  weeks  in  solution  of  Miiller,  and  the  sections  mor- 
danted [for  myelin  stains]  with  0*5  per  cent,  chromic  acid). 

For  special  mixtures  for  GOLGI  impregnations  see  §  752. 

693.  Nervous  Centres  of  Reptiles,  Fishes,  and  Amphibia. — 
MASON  (Central  Nervous  System  of  Certain  Reptiles,  etc.;  WHITMAN'S 
Methods,  p.  196)  recommends  iodised  ajcohol,  six  to  twelve  hours;  3  per 
cent,  bichromate,  with  a  piece  of  camphor  in  the  bottle,  and  to  be  changed 
once  a  fortnight  until  the  hardening  is  sufficient  (six  to  ten  weeks). 

BUBCKHABDT  (Das  Centralnervensystem  von  Protopterus,  Berlin,  1892  ; 


388  CHAPTER    XXX. 

Zeit.  f.  wiss.  MiJc.,  ix,  3,  1893,  p.  347)  recommends  a  liquid  composed  of 
300  parts  of  1  per  cent,  chromic  acid,  10  parts  of  2  per  cent,  osmic  acid,  and 
10  parts  of  concentrated  nitric  acid,  in  which  brains  of  Protopterus  are 
hardened  in  twenty-four  to  forty-eight  hours. 

FISH  (Journ.  of  MorphoL,  x,  1,  1895,  p.  234)  employed  for  the  encephalon 
of  Desmognathus  fusca  a  mixture  of  100  c.c.  of  50  per  cent,  alcohol,  5  c.c. 
of  glacial  acetic  acid,  5  grms.  of  corrosive  sublimate,  and  1  grm.  of  picric 
acid,  fixing  for  twelve  to  twenty-four  hours,  and  passing  through  the  usual 
alcohols. 


Imbedding  and   Cutting. 

694.  The  Methods  of  Imbedding. — The  paraffin  infiltration 
method  can  only  conveniently  used  for  the  smaller  objects  of 
this  class.  Human  spinal  cord  (which  is  quite  at  the  upper 
limit  as  regards  size)  can  be  properly  infiltrated  with  paraffin 
by  taking  the  precaution  of  first  cutting  it  up  into  slices  of 
not  more  that  a  few  millimetres— preferably  not  more  than 
one — in  thickness.  The  largest  objects  of  this  class,  such  as 
entire  hemispheres  of  man,  cannot  be  really  infiltrated  with 
any  known  imbedding  mass  in  any  reasonable  time  ;  and  the 
anatomist  must  be  content  with  simple  superficial  imbedding 
— the  mere  production  of  a  mould  of  imbedding  mass  round 
the  tissues — a  proceeding  which  is  here  of  the  greatest 
service.  For  intermediate  objects — those  whose  size  varies 
between  that  of  a  small  nut  and  a  walnut — it  appears  to  me 
that  they  are  best  treated  by  the  collodion  method,  which  is 
at  once  the  safest,  the  most  convenient,  and  the  most  advan- 
tageous as  regards  the  ulterior  treatment  of  sections. 

Imbedding  is  not  a  necessary  process.  Sections  can  be 
•obtained  from  any  part  of  the  central  nervous  system  with- 
out imbedding.  The  material  should  be  very  well  hardened, 
and  a  suitable  piece  should  be  glued  on  to  a  piece  of  wood  or 
cork  by  means  of  a  rather  thick  solution  of  gum  arabic. 
As  soon  as  it  begins  to  stick  to  the  support  the  whole  is 
thrown  into  80  per  cent,  alcohol  to  harden  the  joint,  after 
which  it  may  be  fixed  in  the  object-holder  of  the  microtome 
and  cut. 

If  the  collodion  method  has  been  taken  a  difficulty  may 
arise.  It  may  be  found  that,  notwithstanding  every  pre- 
caution, the  collodion  has  not  thoroughly  penetrated  the 
tissues.  Good  sections  may,  however,  still  be  obtained  by 


NEUKOLOGICAL    METHODS.  389 

DOVAL'S  method  of  collodionising  the  sections.  The  cut  sur- 
face of  the  tissue  is  dried  by  blowing  on  it,  and  is  covered 
with  a  thin  layer  of  collodion  laid  on  it  with  a  brush.  As 
soon  as  this  layer  has  somewhat  dried,  which  happens  very 
rapidly,  a  section  is  cut  and  the  cut  surface  is  collodionised 
as  before,  and  so  on  for  each  section.  This  process  gives 
very  good  results,  and  may  be  advantageously  employed  even 
with  material  that  has  been  successfully  imbedded,  as  it 
gives  a  better  consistency  to  the  tissue,  and  enables  thinner 
sections  to  be  obtained  (VAN  GEHDCHTEN,  in  litt.). 

STRASSER  (Zeit.  f.  u-iss.  Mik.,  ix,  1892,  p.  8)  obtains  paraffin 
sections  of  10  cm.  breadth  by  15  cm.  length.  He  cuts  out 
from  hardened  material  slices  of  from  1  to  2  cm.  in  thickness, 
de-alcoholises  them  with  xylol-carbolic  acid  mixture,  §  161, 
allows  this  to  evaporate,  and  brings  them  first  into  melted 
yellow  vaselin,  and  lastly  either  into  a  mixture  of  vaselin  and 
paraffin  of  42°  melting  point,  or  into  pure  paraffin. 

He  also  imbeds  the  slices  in  celloidin,  and  clears  them 
before  cutting  with  a  mixture  of  xylol-carbolic  acid  and  80 
per  cent,  alcohol  in  equal  parts. 

FEIST  (Zeit.  f.  iviss.  Mile.,  viii,  4,  1892)  gives  a  useful  hint  for  marking 
the  right  and  left  sides  of  spinal  cord.  He  imbeds  with  each  segment  of 
the  cord  a  small  cylinder  (of  about  1  square  millimetre  in  section)  of  hard- 
ened liver,  stuck  vertically  in  the  imbedding  mass  (either  celloidin  or 
paraffin)  against  the  side  of  the  cord  that  it  is  desired  to  mark. 

For  further  details  concerning  imbedding  and  cutting,  see 
last  edition. 


CYTOLOGICAL  METHODS. 
(a)   Nerve  cells. 

695.  NISSL'S  Methylen-blue  Method  (Neurol  Centralb.,  1894, 
p  508). — Fresh  material  is  hardened  in  96  per  cent,  alcohol, 
and  sectioned  without  imbedding.  The  sections  are  brought 
into  a  watch  glass  with  the  following  stain  : 

Methylen  blue  (Methylenblau  pat.)     .      3' 75  parts. 
Venice  soap    .  .      1*75      „ 

Distilled  water  lOOO'O   ^   „ 

The  watch  glass  is  warmed  over  a  flame  to  about  65°  to  70°  C., 
till  bubbles  are  given  off  which  burst  at  the  surface  of  the 


390  CHAPTER    XXX. 

liquid.  The  sections  are  then  brought  into  a  mixture  of  10 
parts  of  anilin  oil  with  90  parts  of  96  per  cent,  alcohol,,  and 
are  differentiated  therein  until  colour  is  no  longer  given  off 
from  them.  They  are  got  on  to  a  slide,  dried  with  filter- 
paper,  cleared  with  oil  of  cajeput,  dried  again  with  filter- 
paper,  treated  with  a  few  drops  of  benzin,  and  mounted  in 
benzin-colophonium. 

Prof.  VAN  GEHUCHTEN  writes  me  that  he  prefers  to  take 
paraffin  sections,  mounted  on  slides  by  the  water  method 
(§  182),  and  stain  them  for  five  or  six  hours  in  Nissl's  mixture 
in  a  stove  kept  at  .35°  to  40°  C.  Differentiation  is  done  as 
above,  and  the  sections  are  mounted  in  xylol-damar. 

In  a  later  paper  (op.  cit.,  p.  781 ;  Zeit.  f.  wiss.  Mik.,  xiii, 
2,  1896,  p.  237)  NZSSL  advises  that  after  covering  the  sections 
with  a  drop  of  the  benzin-colophonium  they  should  be  passed 
through  a  flame.  The  benzin  gases  ignite,  and  must  be 
blown  out  immediately,  and  the  operation  repeated  until  the 
medium  no  longer  ignites.  This  is  said  to  prevent  diffusion 
of  the  stain  after  mounting. 

Slight  modification  by  TELJATNIK,  Neurol.  Centralb.,  xv,  1896,  p.  1129  ; 
Zeit.  f.  wiss.  Mile.,  xiv,  1897,  p.  79. 

SADOVSKY  (C.  R.  Soc.  Biol.,  iii,  1896,  p.  353)  stains  sections 
of  formol  material  for  a  quarter  of  an  hour  to  several  hours 
in  1  per  cent,  methylen  blue  (or  for  one  to  three  minutes  in 
a  concentrated  solution  of  fuchsin  in  "  5  per  cent,  carbolic 
acid  water"),  then  treats  them  on  the  slide  with  1  per  cent, 
acetic  acid  until  the  grey  substance  is  clearly  differentiated 
from  the  white,  dehydrates  in  absolute  alcohol  and  passes 
through  xylol  into  balsam.  The  fuchsin  gives  the  sharper, 
stain. 

GOTHABD  (op.  cit.,  v,  1898,  p.  530;  Zeit.  f.  wiss.  Mik.,  xv, 
4,  1899,  p.  487)  stains  celloidin  sections  for  twenty- four  hours 
in  Unna's  polychromatic  methylen  blue  and  differentiates  in 
a  mixture  of  5  parts  of  creosote,  4  of  oil  of  cajeput,  5  of 
xylol,  and  16  of  absolute  alcohol. 

LORD  (Journ.  Ment.  Sci.,  Oct.,  1898;  Zeit.  f.  wiss.  Mik.,  xvi, 

1899,  p.  59)  treats  sections  of  fresh  material  frozen  for  a  few 

seconds  before   staining  with    a    mixture   of  equal  parts  of 

,  6  per  cent,  formol  and  saturated  solution  of  picric  acid.     He 


NEUROLOGICAL   METHODS.  391 

clears  with  origanum  oil.  See  also  Jou-rn.  Roy.  Mic.  Soc., 
1899,  p.  448. 

LUITHLEN  and  SORGO  (Neurol.  Centralb.,  xvii,  1898,  p.  640; 
Zeit.  f.  wiss.  Mik.,  xv,  1899,  p.  359)  differentiate  in  Unna's 
glycerin-ether  mixture  (procurable  from  Griibler  &  Hollborn), 
remove  this  with  absolute  alcohol  and  clear  in  origanum  oil. 

EWING  (New  York  Med.  Bee.,  1898,  p.  513;  Zeit.  f.  wiss. 
Mik.,  xvi,  1899,  p.  95)  prefers  to  differentiate  simply  in 
absolute  alcohol,  clear  in  oil  of  cajeput,  and  mount  in  balsam. 

See  also  some  slight  modifications  in  GOLDSCHEIDEE  &  FLATAU,  Nor  male 
und  path.  Anat.  der  Nervenzellen,  etc.,  Berlin,  Kornf eld,  1898 ;  Zeit.  f. 
wiss.  Mik.,  xvi,  1899,  p.  102,  and  NISSL'S  remarks  thereon,  Deutsche  Zeit. 
Nervenheilk.,  xiii,  1899,  p.  348 ;  Zeit.f.  wiss.  Mik.,  xvi,  1899,  p.  370. 

Further,  Cox,  Intern.  Monatsschr.  Anat.  Phys.,  xv,  1898,  Heft  8 ;  Zeit. 
f.  wiss.  Mik.,  xvi,  1899,  p.  101. 

696.  Methylen  Blue   and  Erythrosin.—  HELD    (Arch.  Anat. 
Phys..,  Anat.  Abth.,   1895,   1896,   p.  399)   stains  sections   on 
slides,  with  the  aid  of  a  gentle  heat,  for  one  or  two  minutes 
in  a  solution  of    1  grm.  of    Griibler's   erythrosin    in    150  of 
water  with  two  drops  of  glacial  acetic  acid,  washes  out  with 
water,    and   stains    in   a   mixture   of   equal    parts   of   NissFs 
methylen  blue  and  5  per  cent,  solution  of  acetone,  warming 
strongly  the  while,  until  all  odour  of  acetone  has  disappeared. 
After  cooling  he  differentiates  with  O'l  per  cent,  solution  of 
alum  until  the  sections  appear  reddish,  rinses  in  water,  de- 
hydrates as  rapidly  as  possible  in  absolute  alcohol,  and  passes 
through  xylol  into  balsam.      For  some   slight  modifications 
see   further  HELD,  op.  cit.,  1897,   pp.    226—233,   273—305 
(Supplementband),  and  BOCOARDI,  Mon.  Zool.  Ital.,  x,  1899, 
p.  141  ;   Zeit.  f.  wiss.  Mik.,  xvi,  4,  1900,  p.  471  (stains  in  a 
mixture  of  erythrosin  O'l,  toluidin  blue  0*2,  and  water   100 
parts,  and  differentiates  in  0'5  per  cent,  alum  solution). 

697.  Thionin. — LENHOSSEK    (Fein.    Bau.    d.   Nerve  11 systems, 
Berlin,  1894,  p.  149)  stains  sections  of  formol  material   for 
five  minutes  in  a  concentrated  aqueous  solution  of  thionin, 
rinses  with  water,  differentiates  in  a  mixture  of  1  part  anilin 
oil  to  9  of  absolute  alcohol,  and  passes  through  oil  of  cajeput 
or  xylol  into  damar  or  balsam.    The  stain  does  not  keep  well. 

Similarly  RAMON  Y  CAJAL,  Man.  de  Anat.  Path.  Gen.,  1896 


392  CHAPTER  XXX. 

(see  Zeit.  f.  wiss.  Mik.,  xv,  1899,  p.  375),  and  LUXENBURG, 
Neurol.  Centralb.,  xviii,  1899,  p.  629 ;  Zeit.  f.  wiss.  Milt.,  xvi, 
4,  1900,  p.  477. 

698.  Toluidin  Blue. — LENHOSSEK    (Neurol.    Centralb.,    xvii, 
1898,  p.  577;   Zeit.  f.  wiss.  Mik.,  xv,   1899,  p.  492)  thinks 
that  for  the  study  of  the  corpuscles  of  Nissl  toluidin  blue  is 
preferable.      Sections  are   stained   on    slides  for  a  night  in 
concentrated  solution  of  toluidin  blue,  rinsed  in  water,  quickly 
differentiated  with  alcohol,  cleared  with  xylol  or  carbolic- acid 
xylol,  and  mounted   in  balsam.      They  may  be  very  lightly 
counter-stained  with  erythrosin  before  the  differentiation. 

Similarly  POLUMOEDWINOW  (Zeit.  f.  wiss.  Mik.,  xvi,  1899, 
p.  371),  who  stains  in  a  very  weak  alkaline  solution,  1  part 
of  1  per  cent,  solution  to  119  of  water  and  1  of  carbonate  of 
soda. 

699.  Neutral  Red. — JULIDSBURGEE    (Neurol.    Centralb.,   xvi, 
1897,  p.  259;  Zeit.  f.  wiss.  Mik.,  xiv,  2,  1897,  p.  211)  stains 
sections  of  formol  material  for  half  to  three  quarters  of  a 
minute  in  warm  1  per  cent,  solution  of  neutral  red,  dehy- 
drates in  alcohol,  and  passes  through  bergamot  oil  to  balsam. 

ROSIN  (Deutsche  med.  Wochenschr.,  1898,  No.  39,  p.  615; 
Zeit.  f.  wiss.  MiJc.,  xvi,  2,  1899,  p.  238)  stains  in  concentrated 
aqueous  solution,  washes  out  thoroughly  with  water,  and 
passes  through  alcohol  (must  be  free  from  acid)  into  xylol 
and  balsam.  A  double,  metachromatic  stain,  granules  of 
Nissl  red,  nucleoli  red,  all  the  rest  yellow. 

700.  Methods  of  EEHM  (Munchener  med.  Wochenschr.,  1892,  No.  13 ;. 
Zeit.f.  wiss.  MiJc.,  ix,  3,  1893,  p.  390),  see  last  edition. 

701.  WEIGEKT'S  Stain  for  Nuclear  Figures  (Ally.  Zeit.  f. 
Psychiatric,  1,  1894,  p.  245). — Sections  of  alcohol  material, 
cut  without  imbedding,  are  put  for  half  an  hour  into  Tinct. 
Ferri  Acet.  Rademacheri,  rinsed,  stained  for  a  quarter  of  an 
hour  in  1  per  cent,  solution  of  haBmatoxylin  in  water,  rinsed, 
differentiated  rapidly  in  70  per  cent,  alcohol  containing  1  per 
cent,  of  hydrochloric  acid,  and  mounted  in  balsam. 

702.  Other  Methods.— See  Cox,  Zeit.f.  wiss.  MiJc.,  xiii,  1896,  p.  498  ; 
xv,  3,  p.  369 ;  xvi,  1,  1899,  p.  101  ;  Anat.  Hefte,  xxxi,  1898,  p.  75  }    Intern. 


NEUROLOGICAL   METHODS.  393 

Monatsschr.,  xv,  1898,  H.  8;  AUERBACH,  Monatsschr.  Psychiatric,  iv,  1898, 
p.  31 ;  Zeit.f.  wiss.  Mik.,  xv,  1899,  p.  493;  BUEHLER,  Verh.  Phys.-Med. 
Ges.  Wiirzburg,  xxxi,  1898,  p.  285  ;  Zeit.f.  wiss.  Mik.,  xv,  1899,  p.  351. 


(b)    Nerve  Fibres. 

703.  Structure  of  Medullated  Nerve. — In   order   to  demon- 
strate  the   axis- cylinder  and   the    sheath   of    Schwann,   the 
myelin  may  be  removed.      This  may  be  done  by  boiling  in 
caustic  soda,  and  then  neutralising ;  by  boiling  in  a  mixture 
of  absolute  alcohol  and  ether,  and  adding  caustic  soda ;   by 
boiling  in  glacial  acetic  acid;   by  boiling  in  fuming  nitric 
acid,  and  adding  caustic  potash ;  or  by  treating  with  eau  de 
Javelle ;    or   (VAN   GEHDCHTEN,  in   litt.)  the   myelin   may  be 
extracted  in  the  cold  by  leaving  the  nerves  for  some  time  in 
a  mixture  of  alcohol  and  ether. 

704.  Axis-Cylinder,   KUPFFEK'S    Method   (Sitzb.  math.  phys. 
Kl.  If.  Bayr.  Akad.  Wiss.,  xiii,  1884,  p.  470 ;  Z&it.  f.  wiss.  Mik., 
1885,  p.  106). — A  nerve  is  stretched  on  a  cork  and  treated 
for  twenty-four  hours  with   O5  per  cent,  osmic   acid.      It  is 
then  washed  in  water  for  two  hours  and  stained  for  twenty- 
four  to  twenty- eight  hours  in  saturated  aqueous  solution  of 
Saurefuchsin  ;  after  which  it  is  washed  out  for  from   six  to 
twelve   hours    (not   more   in   any    case)  in   absolute   alcohol, 
cleared  in  clove  oil,  imbedded  in  paraffin,  and  cut. 

Iron  haematoxylin  sometimes  gives  a  sharp  stain  of  axis- 
cylinders. 

See  also  the  complicated  method  of  AUERBACH,  Neurol.  Centralb.,  1897, 
p.  439  ;  Zeit.f.^wiss.  Mik.,  xiv,  1897,  p.  402. 

705.  Formol-Methylen-Blue. — ROSSOLIMOW    and     MUUAWIEW 
(Neurol.  Centralb.,  xvi,  1897,  p.  722;  Zeit.f.  wiss.  Mik.,  xiv, 
1898,  p.  511)  harden  pieces  of  nerve  in  2   per  cent,  formol 
solution   for   two  days,  then   for  two  more  in  4  per  cent., 
tease  or  section,  stain  in  warmed  methylen  blue,  with  heat, 
and    differentiate  with    anilin  alcohol,  as  in  Nissl's  process, 
clear  in  oil  of  cajeput,  and  mount  in  balsam. 

706.  Neuroceratin  Structures  (&ALLI,  Zeit.f.  wiss.  Mik.,  iii,  1, 1886, 
p.  467). — Small  portions  of  ischiatic  nerve  are  treated  for  one  or  two  days 


394  CHAPTER   XXX. 

with  solution  of  Miiller  diluted  with  2  parts  of  water,  then  for  a  quarter 
of  an  hour  with  glycerin  containing  1  or  two  drops  of  glacial  acetic  acid 
for  each  cubic  centimetre,  and  finally  (without  previous  washing  with 
water)  are  stained  for  fifteen  to  twenty  minutes  in  aqueous  solution  of 
Ohina  blue,  washed  out  in  alcohol,  cleared  in  essence  of  turpentine,  and 
mounted  in  damar. 

PLATNEE'S  Method  (Zeit.  f.  wiss.  Mik.,  vi,  2,  1889,  p.  186). — Small 
nerves  are  fixed  and  hardened  for  several  days  in  a  mixture  of  1  part  of 
Liq.  Ferri  Perchlor.  (Ph.  G.,  ed.  2)  and  3  to  4  parts  of  water  or  alcohol, 
washed  out  in  water  or  alcohol  till  no  traces  of  iron  remain  in  them, 
stained  for  several  days  or  weeks  in  a  concentrated  solution  of  "  Echtgriin  " 
in  75  per  cent,  alcohol,  dehydrated,  imbedded,  and  sectioned.  See  also 
BEEE,  Jahrb.  Psychiatric,  ii,  1893,  1  Heft. 

See  also  the  papers  of  GEDOELST  in  La  Cellule,  iii,  1887,  p.  117,  and  v, 
1889,  p.  126  (good  details  of  digestion  methods)  ;  also  the  report  in  Zeit.  f. 
wiss.  Mik.,  vii,  1,  1890,  p.  57. 

Cox  (Anat.  Hefte,  i,  H.  31,  1898,  p.  75  ;  Zeit.  f.  wiss.  Mik.,  xv,  1899, 
p.  369)  fixes  nerves  in  osmic  acid  of  2  per  cent,  (rabbit)  or  1  per  cent, 
(frog),  washes,  dehydrates,  clears  with  bergamot  oil,  and  mounts  in  balsam. 
The  bergamot  oil  dissolves  out  the  myelin,  and  leaves  the  neuroceratin 
visible.  It  may  be  necessary  to  leave  the  nerves  for  forty-eight  hours  in 
the  oil. 


707.  APATHY'S  Neurofibrils  (APATHY,  Mitth.  ZooL  Stat. 
Neapel,  xii,  1897,  p.  712),—  The  Hsematein  Method.— 
Material  may  be  fixed  with  sublimate,  liquid  of  Zenker, 
picro-sulphuric  acid,  or  any  mixture  that  is  not  inimical  to 
staining  with  alum  haematoxylin,  and  should  be  preserved  in 
90  per  cent,  alcohol.  Portions  are  stained  for  at  least  forty- 
eight  hours  in  the  haematein  solution  I  A,  §  247,  and  are 
then  washed  for  up  to  twenty-four  hours  in  absolutely  pure 
distilled  water,  preferably  suspended  therein.  Before  the 
stain  has  become  washed  out  of  the  neurofibrils,  it  is  fixed 
therein  by  putting  the  preparations  for  three  to  five  hours 
into  spring  water,  after  which  they  are  put  back  for  not 
more  than  two  hours  into  distilled  water,  dehydrated  as 
rapidly  as  possible  by  hanging  them  up  in  absolute  alcohol, 
and  imbedded  in  paraffin,  or  celloidin,  or  glycerin  jelly; 
they  must  be  protected  from  the  light  whilst  in  the  chloro- 
form through  which  they  are  passed  into  the  paraffin,  or 
whilst  in  the  celloidin.  Sections  are  made  and  mounted  in 
a  resin  or  in  neutral  glycerin. 

APATHY'S    Gold   Chloride  After-gilding  Method    has    been 
given,  §  358 ;  his  Fore-gilding  Method,  §  355,  p.  255. 


NEUROLOGICAL   METHODS.  395 

See  also  BETHE  (Arch.  f.  mik.  Anat.,  li,  1898,  p.  385),  where  are  also 
some  details  as  to  staining  with  methylen  blue,  and  a  new  method  with 
toluidin  blue  after  mordanting  with  molybdic  acid. 

708.  Other  Methods  for  Medullated  Nerve. — RANVIEB,  Traite, 
p.  718,  et  seq.;  REZZONICO,  Arch,  per  le  Sci.  Med.,  1879,  p.  237  ;  TIZZONI, 
ibid.,  1878,  p.  4  (a  process  of  boiling  in  chloroform  for  an  hour  or  two,  then 
staining  and  mounting  in  glycerin)  ;  BOVEEI,  Zeit.  f.  wiss.  Mik.,  iv,  1, 
1887,  p.  91 ;  JAKIMOVITCH,  Journ.  de  I'Anat.,  xxiii,  1888,  p.  142,  or 
Zeit.  /.  wiss.  Mik.t  v,  4,  1888,  p.  526  (instructions  for  impregnating  the 
axis-cylinder  with  silver,  followed  by  reduction  in  formic  acid  and  amyl 
alcohol) ;  SCHIEFFEBDECKEB,  in  BEHBENS,  KOSSEL,  u.  SCHIEFFEBDECKEB, 
Das  Mikroskop,  Bd.  ii,  p.  227  ;  HUBEE,  Zeit.  f.  wiss.  Mik.,  x,  3,  1893, 
p.  394  (stains  with  BENDA'S  safranin  and  Lichtgriin) ;  EABL,  ibid.,  xi,  1, 
1894,  |>.  42  (the  lines  of  Frommann  are  artefacts  due  to  the  silver  nitrate)  ; 
FISCHEL,  ibid.,  p.  48  (similar  conclusion) ;  TIBELLI,  ibid.,  xi,  3,  1894, 
p.  391 ;  SEGALL,  Journ.  de  I'Anat.,  xxix,  1893,  p.  586 ;  MABCHESINI, 
Anat.  Anz.,  xii,  1896,  p.  211  (sublimate  and  sulphide  of  potassium). 


CHAPTER  XXXI. 
NEUROLOGICAL    METHODS — NERVE-FIBRE     STAINS     (WEIGERT 

AND  OTHERS). 

a.  Myelin  Stains. 

709.  Introduction. — The  most  important  of  the  methods  for 
the  study  of  tracts  of  medullated  nerve-fibres  are  the  haema- 
toxylin  methods  of  WEIGEET. 

There  have  been  in  all  three  methods  of  WEIGERT  ;  the 
1884  method,  the  1885  method,  and  the  1891  method.  The 
ordinary  methods  of  staining  with  haematoxylin  depend  on 
the  employment  of  an  aluminium  lake  of  haamatoxylin. 
WEIGKRT'S  method  depends  on  the  formation  of  another  lake, 
a  chromium  or  copper  lake.  In  consequence  of  the  forma- 
tion of  these  lakes  haematoxylin  acquires  the  property  of 
staining  the  myelin  of  nerves  in  a  quite  specific  way. 

In  WEIGEUT'S  process  the  formation  of  these  lakes  takes 
place  in  the  tissue  itself.  The  details  of  the  process  have 
been  considerably  modified,  both  by  other  workers  and  by 
WEIGERT  himself.  The  1884  method  (Fortschr.  d.  Med.,  1884, 
pp.  113,  190;  Zeit.  f.  wiss.  Mik.,  1884,  pp.  290,  564),  which 
depends  on  the  formation  of  a  chrome  lake,  may  be  considered 
to  be  superseded.  Not  so  the  two  others,  which  depend  on 
the  formation  of  a  copper  lake. 

For  a  critical  history  of  these  methods,  see  WEIGERT,  in 
Ergebnisse  der  Anatomie,  vi,  1896  (1897),  p.  5. 

710.  WEIGKRT'S  1885  Method  (Fortschr.  d.  Med.,  1885,  p. 
136;  Zeit.f.  wiss.  Mi'k.,  1885,  pp.  399,  484;  Ergebnisse  der 
Anatomie,  vi,  1896  [1897],  p.  10).— The  tissues  are  to  be 
hardened  in  bichromate  of  potash.  WEIGERT  takes  (Ergeb- 
nisse,  p.  10)  a  5  per  cent,  solution,  and  if  time  is  an  object 


NEUROLOGICAL    METHODS.  397 

hardens  in  a  stove.  (Other  bichromate  mixtures  will  do, 
e.  g.  Miiller's,  Kultschizky's,  Zenker's  ;  Erlicki's  is  not  to  be 
recommended).  The  tissues  are  "  ripe  "  for  staining  when 
the  hardening  has  been  carried  to  a  certain  point.  They 
are  first  (Ergebnisse,  p.  13)  yellow,  without  differentiation  of 
the  grey  matter  from  the  white  ;  these  are  unripe.  Later 
they  show  the  grey  matter  light  brown,  the  white  matter 
dark  brown  (owing  to  reduction  of  a  part  of  the  bichromate 
to  a  chrome  oxide  in  the  medullary  sheaths)  ;  these  are 
"  ripe."  If  the  hardening  be  continued  "  all  the  more 
highly  oxidised  chrome  will  pass  into  the  lower  stage  of  oxida- 
tion, and  the  tissues  will  become  green. "  The  tissues  are 
then  over-ripe,  and  cannot  be  used  for  myelin-staining  without 
mordanting  with  copper  or  the  like. 

After  due  hardening,  the  preparation  is  imbedded  by  in- 
filtration with  celloidin  (if  desired  :  imbedding  is  not  obliga- 
tory) and  the  celloidin  block  fastened  on  cork  and  hardened 
in  the  usual  way.  The  hardened  block  is  put  for  one  or 
two  days  into  saturated  solution  of  neutral  acetate  of  copper 
diluted  with  one  volume  of  water,  the  whole  being  kept  at  the 
temperature  of  an  incubating  stove.  By  this  treatment  the 
tissues  become  green  and  the  celloidin  bluish  green.  The 
mordantage  of  the  tissues  is  now  terminated,  and  the  pre- 
paration may  ba  kept  till  wanted  for  sectioning  in  80  per 
cent,  alcohol. 

Sections  are  made  with  a  knife  wetted  with  alcohol,  and 
are  brought  into  a  stain  composed  of — 

Haematoxylin   .  .  .  0*75  to  1  part. 

Alcohol  ......       10  parts. 

Water 90     „ 

Saturated  solution  of  lithium  carbonate        1  part. 

(A  trace  of  any  other  alkali  may  be  added  in  the  place  of  lithium  car- 
bonate. The  object  of  adding  a  little  of  some  base  is  to  "  ripen  "  the 
haematoxylin  solution  ;  or  the  solution  may  be  made  up  with  hsematein, 
and  the  alkali  omitted.) 

The  sections  remain  in  the  stain  for  a  length  of  time  that 
varies  according  to  the  nature  of  the  tissues  :  spinal  cord, 
two  hours  ;  medullary  layers  of  brain,  two  hours  ;  cortical 
layers,  twenty-four  hours. 

They  are  then  rinsed  with  water,  and  brought  into  a  de- 
colourising solution  composed  of — 


398  CHAPTEE    XXXI. 

Borax      ......      2'0  parts. 

Ferricyanide  of  potassium  .  .      2'5      „ 

Water    .  .  .200*0      „ 

They  remain  in  the  solution  until  they  are  decoloured  to 
the  right  degree — that  is,  until  complete  differentiation  of 
the  nerves  (half  an  hour  to  several  hours),  and  are  then 
rinsed  with  water,  dehydrated  with  alcohol,  and  mounted  in 
balsam.  They  may  be  previously  stained,  if  desired,  with 
alum-carmine  for  the  demonstration  of  nuclei. 

The  results  are  most  splendid.  The  blue-black  nerves 
stand  out  with  admirable  boldness  on  a  golden  ground.  The 
method  is  applicable  to  the  study  of  peripheral  nerves  as 
well  as  to  nerve  centres,  and  is  likely  to  be  of  utility  in 
Vertebrate  embryology. 

Nerve  tissue  is  not  the  only  tissue  stained  by  the  process, 
which  can  be  usefully  applied  to  lymphatic  glands  and  to 
skin  (see  SCHTEFFEKDECKER,  in  Anat.  Anz.,  ii,  1887,  p.  680). 

The  process  is  applicable  to  tissues  that  have  been  hardened  in  alcohol  or 
in  any  other  way,  provided  that  they  be  put  into  a  solution  of  a  chromic 
salt  until  they  become  brown,  before  mordanting  them  in  the  copper  solu- 
tion. 

It  is  not  necessary  that  the  mordantage  be  done  in  bulk.  MAX  FLESCH 
(Zeit.  f.  wiss.  MiJc.,  iii,  1,  1886,  p.  50)  prefers  (following  LICHTHEIM)  to 
make  the  sections  first,  and,  after  mordanting,  bring  them  on  a  spatula  into 
70  per  cent,  alcohol,  and  thence  into  the  stain. 

For  a  method  for  regenerating  the  staining  solution  after  use,  see  FANNY 
BEELINEEBLAU,  Zeit.f.  wiss.  Mile.,  1886,  p.  50,  or  previous  editions. 

PANETH  (ibid.,  1887,  p.  213)  makes  the  stain  with  extract  of  logwood 
instead  of  pure  hsematoxylin. 

BEEGLIA  (ibid.,  vii,  2,  1890,  p.  236;  see  also  Journ.  Roy.  Mic.  Soc.,  1890, 
p.  817)  stains  with  liquid  extract  of  logwood  or  Pernambuco  wood. 

For  both  of  these  see  previous  editions. 

GEEOTA  (Intern.  Monatsschr.  Anat.,  xiii,  1896,  pp.  138,  139 ;  Zeit.  f. 
wiss.  Mik.,  xiii,  3,  1896,  p.  315)  states  that  the  reaction  can  be  obtained 
by  using  the  copper  after  the  stain,  and  that  an  alum-haematoxylin  may  be 
used.  He  dissolves  6  grm.  haematoxylin  in  60  of  absolute  alcohol,  and 
adds  200  of  1  per  cent,  alum  solution,  lets  the  solution  stand  for  ten  days, 
stains  therein  sections  for  four  to  twenty-four  hours  at  37°  C.,  puts  them 
for  2  hours  into  the  copper  acetate  at  37°  C.,  and  then  differentiates. 


711.  WEIGEET'S  1891  Method  (Deutsche  med.   Wochenschr., 
42,  1891,  p.  1184;   Zeit.f.  wiss.  Mik.,  viii,  3,  1891,  p.  392).— 


NEUROLOGICAL    METHODS.  399 

Tht1  material  is  to  be  hardened  in  bichromate  and  imbedded 
in  celloidin  in  the  usual  way.  The  hardened  blocks  of  cel- 
loidin  are  brought  into  a  mixture  of  equal  parts  of  a  cold 
saturated  solution  of  neutral  acetate  of  copper  and  10  per  cent, 
aqueous  solution  of  potassio-tartrate  of  sodium  (C4H406KNa 
+  4HoO,  salt  of  Seignette).  They  are  left  in  the  mixture  for 
twenty-four  hours  in  an  incubator.  (Large  specimens  [pons] 
will  require  forty-eight  hours,  the  mixture  being  changed  for 
fresh  at  the  end  of  twenty -four  hours.)  They  are  then 
brought  for  twenty-four  hours  into  aqueous  solution  of 
neutral  acetate  of  copper,  either  saturated  or  diluted  with  1 
volume  of  water,  being  kept  as  before  in  the  incubator. 
They  are  then  rinsed  with  water  and  brought  into  80  per 
cent,  alcohol,  in  which  they  may  either  remain  till  wanted  or 
be  cut  after  half  an  hour. 

Sections  are  made  and  stained  for  from  four  to  twenty-four 
hours  at  the  temperature  of  the  room  in  a  freshly  prepared 
mixture  of  9  vols.  of  (A)  a  mixture  of  7  c.c.  of  saturated 
aqueous  solution  of  carbonate  of  lithium  with  93  c.c.  of  water, 
and  1  vol.  of  (B)  a  solution  of  1  grm.  of  haematoxylin  in.  10 
c.c.  of  alcohol  (A  and  B  may  be  kept  in  stock,  but  A  must  not 
be  too  old).  The  sections  should  be  loose  ones,  not  such  as 
have  been  seriated  in  celloidin,  and  should  not  be  thicker 
than  0*025  mm.  The  stain  is  poured  off  and  the  sections 
are  washed  in  several  changes  of  water  poured  on  to  them. 
They  are  then  treated  with  90  per  cent,  alcohol,  followed  by 
carbolic -acid-and-xylol  mixture  (for  a  short  time  only),  or  by 
a  mixture  of  2  parts  of  anilin  oil  with  1  of  xylol,  then  pure 
xylol  and  xylol  balsam  (not  chloroform  balsam,  which  injures 
the  stain). 

Medullated  fibres  dark  blue  on  a  light,  sometimes  rosjr 
ground.  If  it  be  wished  to  have  the  ground  particularly 
colourless,  take  instead  of  the  second  wash-water  a  mixture 
of  ±  to  J  volume  of  common  (not  glacial)  acetic  acid  with  100 
volumes  of  water.  Thick  sections  or  series  in  celloidin  re- 
quire a  special  differentiation.  They  may  be  differentiated 
either  with  the  above-mentioned  acetic  acid  mixture,  or  in 
the  usual  borax-ferricyanide  mixture  diluted  with  water.  In 
the  latter  case  the  ground  will  be  yellow. 

If  the  impregnation  with  the  copper  be  imperfect  (as,  for 
instance,  may  happen  if  the  treatment   with  the  copper   salt 


400  CHAPTER   XXX L. 

"be  performed  at  the  normal  temperature  instead  of  in  an  in- 
cubator) some  instructive  differentiations  of  ganglion- cells 
may  be  obtained,  the  processes  of  the  cells  of  Purkinje  in  the 
cerebellum,  for  instance,  being  very  sharply  brought  out ; 
but  such  preparations  have  a  tendency  to  after-blackening, 
which  does  not  happen  with  those  that  have  been  thoroughly 
impregnated  with  the  copper. 

The  advantages  of  the  improved  method  are  that  differen- 
tiation after  staining  is  not  necessary;  that  the  annoying 
precipitates  formed  on  the  surface  of  the  preparations  by  the 
copper  in  the  old  method  do  not  appear ;  that  the  divers 
manipulations  are  simpler  and  easier;  the  preparations  are 
equal  in  beauty  to  those  of  Pal,  and  can  be  obtained  with 
greater  certainty.  But  it  is  not  so  well  applicable  to  series 
of  sections  by  WEIGERT'S  Collodion  Method,  §  195,  because 
the  sections  must  be  thin. 

Since  th'e  first  publication  of  this  method,  it  has  been 
discovered  (WEIGERT,  Ergebnisse  der  Anat.,  iii,  1894,  p.  21) 
that  preparations  made  as  above,  without  differentiation  in 
the  ferricyanide  liquid,  do  not  keep  well.  Weigert  therefore 
now  advises  that  they  be  mordanted  as  above  with  salt  of 
Seignette,  which  has  the  advantage  of  preventing  the  forma- 
tion of  precipitates  on  the  surface  of  the  preparations,  but 
that  they  be  also  differentiated  in  the  ferricyanide,  as  in  the 
1885  method. 


Modifications  of  Weigert's  Method. 

712.  PAL'S  Method  (Wien.  med.  Jahrb.,  1886;  Zeit.  f.  wiss. 
Mik.,  iv,  1,  1887,  p.  92  ;  Med.  Jahrb.,  1887,  p.  589;  Zeit.  f. 
wiss.  Mik.,  1888,  p.  88).— This  is  a  chrome-lake  process. 
You  proceed  at  first  as  in  WEIGERT'S  process,  but  omitting 
the  copper  bath,  and  you  stain  as  in  WEIGERT'S  process. 
After  staining  in  the  haematoxylin  solution  the  sections  are 
washed  in  water  (if  they  are  not  stained  of  a  deep  blue  a 
trace  of  lithium  carbonate  must  be  added  to  the  water). 
They  are  then  brought  for  twenty  to  thirty  seconds  into 
0'25  per  cent,  solution  of  permanganate  of  potash,  rinsed  in 
water,  and  brought  into  a  decolouring  solution  composed 
of — 


NEUROLOGICAL    METHODS.  401 

Acid.  Oxalic,  pur.       .  .  .  .          1*0 

Potassium  Sulphite*  (Kalium  Sulfuro- 

sum  [S03K2])    .           .           .           .          1-0 
Aq.  Best 200'0 

In  a  few  seconds  the  grey  substance  of  the  sections  is 
decolourised,  the  white  matter  remaining  blue.  The  sections 
should  now  be  well  washed  out,  and  may  be  double-stained 
with  Magdala  red  or  eosin,  or  (better)  with  picro-carmine  or 
acetic- acid-carmine. 

For  further  details  as  to  the  somewhat  elaborate  minutiae 
of  the  process  see  the  papers  quoted,  or  BEHRENS,  KossELr 
and  SCHIEFFERDECKER'S  Das  MikrosJcop,  i,  p.  199. 

PAL'S  process  gives  more  brilliant  results  than  that  of 
Weigert,  the  ground  of  the  preparations  being  totally  colour- 
Jess.  But  it  has  a  defect ;  it  is  less  certain,  or,  to  put  it  in 
another  way,  less  easy  to  control.  The  differentiation  is 
more  energetic  and  rapid  than  is  desirable.  The  whole 
process  of  differentiation  only  lasts  some  seconds  ;  evidently,, 
then,  nn  error  of  judgment  of  only  a  few  seconds  may 
entirely  vitiate  the  result. 

WEIGERT  (f/rgebnisne,  vi,  p.  21)  considers  that  for  very 
thick  sections  the  process  is  superior  to  his  own.  But  it  is 
not  so  safe  for  very  fine  fibres,  and  is  not  applicable  to  his 
collodion  series  method ;  each  section  must  be  treated  sepa- 
rately. 

MAECUS  stains  by  the  Pal  method  sections  of  material  hardened  in- 
formalin,  as  described  §  692. 

See  also  MARINA,  §  692. 

GUDDEN  (Neurol.  Centralb.,  xvi,  1897,  p.  24)  makes  celloidin  sections  of 
material  hardened  in  5 — 10  per  cent,  formol  followed  by  alcohol,  treats 
them  for  ten  hours  with  0'55  per  cent,  chromic  acid,  rinses  with  water,  and 
treats  with  80  per  cent,  alcohol,  then  stains  by  the  method  of  Pal,  adding 
to  the  haematoxylin  a  few  drops  of  dilute  nitric  acid  (MiNNicn). 

TSCHEENYSCHEW  and  KABUSIN  (Zeii.  f.  wiss.  Mik.,  xiii,  1896,  p.  354), 
stain  for  twenty-four  hours  in  the  haematoxylin  of  KULTSCHITZKY. 

DOLLKEN  (Zeit.f.  wiss.  Mik.,  xv,  4,  1899,  p.  444)  stains  for  four  or  five 
days  in  the  haematoxylin  cold,  then  for  two  hours  at  37°  C.,  washes  for  six 
to  eight  hours  in  spring  water,  and  for  a  quarter  of  an  hour  in  distilled 
water  containing  2  to  3  drops  of  caustic  potash  per  litre,  differentiates  in 
the  permanganate  until  the  undeveloped  non-medullated  tracts  (the  method 

*  Not  "  sulphide,"  as  erroneously  given  in  MEBCIEB'S  Les  Coupes  de 
Systeme  Nerveux  Central,  p.  190. 

26 


402  CHAPTER   XXXI. 

is  for  young  animals)  begin  to  appear,  washes  in  distilled  water,  and  puts 
into  1  per  cent,  oxalic  acid  until  the  non-medullated  tracts  appear  light 
brown,  cortex  and  nuclei  darker.  Staining  with  carmine,  etc.,  is  not 
necessary. 

713.  KAISEE  (Neurol.  Centralb.,  xii,  1893,  No.  11,  pp.  364,  368;  Zeit. 
f.  wiss.  Mik.,  xi,  2,  1894,  p.  249)  hardens  first  in  liquid  of  Miiller,  then  for 
eight  days  in  liquid  of  MARCHI  (§  719),  mordants  sections  with  sesqui- 
chloride  of  iron,  stains,  and  differentiates  with  Pal's  liquid.  For  details 
see  previous  editions. 

Similarly  BOLTON  (Journ.  of  Anat.  and  Phys.,  xxxii,  1898,  p.  245  ; 
Zeit.  f.  wiss.  MiJc.,  xv,  4,  1899,  p.  457),  who  makes  sections  of  formalin 
material,  and  mordants  them  for  a  few  minutes  in  1  per  cent,  osniic  acid, 
or  for  a  few  hours  in  iron-alum  or  ammonium  tnolybdate,  stains  in  KULT- 
SCHITZKY'S  hsematoxylin  (next  §),  and  differentiates  by  Pal's  process. 

Similarly  to  this,  LASLETT  (Lancet,  1898,  p.  321;  Journ.  Hoy.  Mic. 
Soc.,  1898,  p.  600),  who  mordants  in  liquid  of  Marchi  (1  week),  makes 
sections,  stains  by  KULTSCHITZKY'S  method,  and  differentiates  by  PAL'S. 

714.  KULTSCHITZKY  (Anat.  Anz.,  1889,  p.  223,  and  1890, 
p.  519  ;  Zeit.  f.  wiss.  Mik.,  vi,  2,  1889,  p.  196,  and  vii,  3,  1890, 
p.  367)  has  given  two  modifications  of  WEIGERT'S  method,  of 
which  the  following  is  the  later  : — Specimens  are  hardened 
for  one  or  two  months  in  solution  of  ErlicM,  imbedded  in 
celloidin  or  photoxylin,  and  cut.  Sections  are  stained  for 
from  one  to  three  hours,  or  as  much  as  twenty-four,  in  a  stain 
made  by  adding  1  grm.  of  hsematoxylin  dissolved  in  a  little 
alcohol  to  100  c.c.  of  2  per  cent,  acetic  acid.  They  are 
washed  out  in  saturated  solution  of  carbonate  of  lithia  or 
soda. 

Differentiation  is  not  necessary,  but  by  adding  to  the  car- 
bonate of  lithia  solution  10  per  cent,  of  a  1  per  cent,  solu- 
tion of  red  prussiate  of  potash,  and  decolourising  therein  for 
two  or  three  hours  or  more,  a  sharper  stain  is  obtained. 
After  this  the  sections  are  well  washed  in  water  and  mounted 
in  balsam. 

WOLTEES  (Zeit.  f.  wiss.  Mile.,  vii,  4,  1891,  p.  466)  proceeds  as  Kult- 
schitzky,  except  that  he  stains  in  a  solution  kept  warm  by  placing  it  on  the 
top  of  a  stove  kept  at  45°  C.  for  twenty-four  hours,  after  which  time  the 
sections  are  dipped  in  solution  of  Miiller,  and  differentiated  by  the  method 
of  Pal. 

KAES  (ibid.,  viii,  3,  1891,  p.  388 ;  Neurol.  Centralb.,  1891,  No.  15)  modi- 
fies  this  by  staining  for  as  much  as  two  or  three  days,  and  performing  the 
differentiation  several  times  over.  It  appears  doubtful  whether  either  of 
these  modifications  is  an  improvement. 


NEUROLOGICAL    METHODS.  403 

715.  MITBOPHANOW  (Zeit.  f.  wiss.  Mile.,  xiii,  1896,  p.  361) 
mordants  photoxylin  sections  for  at  least  twenty-four  hours 
at  40°  C.  in  a  mixture  of  equal  parts  of  saturated  aqueous 
solution  of  acetate  of  copper  and  90  per  cent,  alcohol,  stains 
for  ten  minutes  in  Kultschitzky's  haematoxylin,  and  differenti- 
ates with  Weigert's  ferricyanide.  Or  after  the  copper  bath 
he  stains  for  ten  minutes  in  a  solution  of  1  grm.  haematoxylin 
in  400  c.c.  of  absolute  alcohol  with  4  c.c.  of  acetic  acid,  brings 
into  j  per  cent,  solution  of  cyanide  of  potassium  in  45  per 
cent,  alcohol  until  the  photoxylin  is  discoloured,  then  into 
the  same  with  addition  of  1  per  cent,  solution  of  red  prussiate 
of  potash  until  the  muscles  are  discoloured  (this  refers  to 
sections  through  the  head  of  Anguilla). 


716.  BERKLEY'S  Rapid  Method  (N  enrol.  Centralb.,  xi,  9, 
1892,  p.  270;  Zeit.  f.  wise.  Mile.,  x,  3,  1893,  p.  370).— 
Slices  of  tissue  of  not  more  than  two  and  a  half  millimetres 
in  thickness  are  hardened  for  twenty-four  to  thirty  hours  in 
mixture  of  Flemming ,  at  a  temperature  of  25°  C.,  then  in 
absolute  alcohol,  then  imbedded  in  celloidin  and  cut.  After 
washing  in  water  the  sections  are  put  overnight  into  a  satu- 
rated solution  of  acetate  of  copper  (or  they  may  be  simply 
warmed  therein  to  35°  to  40°  C.  for  half  an  hour).  They 
are  then  washed,  and  stained  for  fifteen  to  twenty  minutes 
in  the  fluid  given  below,  warmed  to  40°  C.,  allowed  to  cool, 
and  differentiated  for  one  to  three  minutes  in  Weigert's 
ferricyanide  liquid,  which  may  be  diluted  if  desired  with 
one  third  of  water.  Water,  alcohol,  bergamot  oil,  xylol- 
balsam. 

The  stain  is  made  as  follows  :  2  c.c.  of  saturated  solution  of 
carbonate  of  lithia  are  added  to  50  c.c.  of  boiling  water  and 
the  solution  boiled  for  two  minutes  more,  when  l\  to  2  c.c. 
of  10  per  cent,  solution  of  haematoxylin  in  absolute  alcohol 
are  added. 

This  method  is  most  suited  to  fresh  material,  and  does 
not  give  good  results  with  tissues  that  have  suffered  post- 
mortem changes.  It  suffers  from  the  defective  penetration 
of  the  liquid  of  Flemming. 

Liquid  of  Flemming   had  been   used  before   by  FBIEDMA.NN  (Neurol. 
Centralb.,  1885). 


404  CHAPTER    XXXI. 

717.  Other  Modifications  or  Similar  Methods.— FLECHSIG,  Arch, 
f.  Anat.  u.  Phys.,  Phys.  Abtli.,  1889,  p.  537 ;  Zeit.f.  wiss.  Mik.,  vii,  1890, 
p.  71;  Journ.  Boy.  Mic.  Soc.,  1890,  p.  538  ;  BEEGLIA,  Zeit.,  vii,  2,  1890, 
p.  36 ;  Rossi,  ibid.,  vi,  2,  188,  1889,  p.  182 ;  MEECIEE,  ibid.,  vii,  4,  1891, 
p.  480;  HAUG,  ibid.,  vii,  2,  1890,  p.  153;  WALSEM,  ibid.,  xi,  2,  1894, 
p.  236;  ROBEETSON,  ibid.,  xiv,  1897,  p.  80  (Brit.  Med.  Journ.,  1897, 
p.  651) ;  HILL,  Brain,  Ixxiii,  1896 ;  Phil.  Trans.,  184u,  1894,  p.  399  ; 
AEONSON,  Centralb.  med.  Wiss.,  1890;  Ergebnisse  der  Anat.,  vi,  1896 
(1897),  p.  22  (differentiates  in  a  watch -glassful  of  water  containing  a  few 
drops  of  a  mixture  of  equal  parts  of  concentrated  solution  of  calcium 
chloride  and  water)  ;  BOEHM  u.  OPPEL,  ibid.,  p.  23,  who  use  in  the  same 
way  hypochlorite  of  sodium  ;  HAEEIS,  Philadelphia  Med.  Journ.,  May 
14th,  1898  (stains  sections  [of  material  hardened  as  for  Weigert's  stain]  for 
several  hours  in  a  1  per  cent,  solution  of  toluidin  blue  in  1  per  cent,  borax 
solution,  and  differentiates  in  saturated  aqueous  solution  of  tannic  acid). 


Other  Myelin  Stains. 

718.  Osmic  Acid  (EXNEE,  Sitzb.  fc.  Akad.  Wiss.  Wien,  1881,  Ixxxiii, 
3  Abth.,  p.  151 ;  BEVAN  LEWIS,  The  Human  Brain,  p.  105). — A  small 
portion  of  brain,  not  exceeding  a  cubic  centimetre  in  size,  is  placed  in  ten 
times  its  volume  of  1  per  cent,  osmic  acid,  replaced  by  fresh  after  two  days. 
In  from  five  to  ten  days  it  is  imbedded  and  cut.  The  sections  are  treated 
by  caustic  ammonia  (20  drops  to  50  c.c.  of  water),  which  clears  up  the 
general  mass  of  the  brain  substance,  leaving  medullated  fibres  black,  and 
are  examined  in  glycerin.  According  to  Weigert  the  method  shows  very 
fine  fibres  indeed.  The  preparations  are  not  permanent. 

719,  MARCHI'S  Method  (for  Degenerate  Nerves)  (Rivista 
sperim.  di  Freniatria  e  di  Med.  leg  ale,  1887,  p.  208  ;  Zeit.  f. 
wiss.  Mik.,  ix,  3,  1893,  p.  350). — Nerves  are  first  hardened 
for  a  week  in  solution  of  Miiller,  and  then  put  for  a  few 
days  into  a  mixture  of  2  parts  solution  of  Miiller  and  1  part 
1  per  cent,  osmic  acid  solution.  The  treatment  with  the 
chrome  salt  deprives  the  medullary  sheath  of  normal  fibres 
of  the  faculty  of  impregnating  with  osmium,  whilst  the 
(fatty)  degeneration  products,  in  diseased  sheaths  retain  that 
faculty.  In  consequence  the  sheaths  in  normal  nerves 
acquire  a  yellow  coloration,  those  of  degenerated  tracts  a 
black  one. 

For  the  study  of  degenerate  nerve-tracts  the  method  of 
MAECHI  has  an  advantage  over  that  of  WEIGERT,  in  that  it 
gives  positive  images  of  the  degenerated  elements,  Weigert's 
process  only  giving  negative  ones. 


NEUROLOGICAL    METHODS.  405 

For  a  critical  review  of  this  method  and  its  modifications 
see  WEJGERT,  in  Eryebnixse  der  Anatomie,  vii,  1897  (1898), 
pp.  1—8. 

The  method  has  been  applied  to  tissues  that  have  been  hardened  in 
formol ;  but  this  (WEIGEBT,  loc.  cit.)  does  not  seem  recommendable. 

VASSALE  (Arch.  Ital  BioL,  xxiv,  1895,  p.  89;  Zeit.  f.  wiss.  Mile.,  xiii, 
4,  1896,  p.  495)  modifies  the  fluid  by  taking  1  per  cent,  osmic  acid  one 
part,  and  three  parts  liquid  of  Miiller,  and  adding  twenty  drops  of  nitric 
acid  to  100  c.c.  of  the  mixture,  but  only  advises  the  modification  for  large 
specimens  which  have  been  a  long  time,  four  to  five  months,  in  the  liquid 
of  Muller. 

FINOTH  (Virchow's  Arch.,  cxliii,  1896,  p.  133;  Zeit.  f. 
wiss.  Mik.,  xiii,  1896,  p.  237)  makes  sections  of  material 
that  has  been  in  liquid  of  Muller  for  not  more  than  a  few 
weeks  or  months,  and  puts  them  for  four  to  ten  hours  into  a 
freshly  prepared  mixture  of  one  or  two  parts  of  1  per  cent, 
osmic  acid  and  one  part  of  a  concentrated  solution  of  picric 
acid  in  one  third  alcohol  (the  mixture  must  be  protected 
from  light  during  the  reaction).  For  peripheral  nerves, 
myelin  (normal),  black. 

BUSCH  (Neurol.  Centralb.,  xvii,  1898,  p.  476  ;  Zeit.  /. 
•wiss.  Mik.,  xv,  1899,  p.  373)  puts  formol-hardened  material 
for  five  to  seven  days  into  a  solution  of  one  part  osmic  acid, 
three  of  iodate  of  sodium,  and  300  of  water.  Same  stain  as 
Marches,  but  more  penetrating  and  sharper. 

TELJATNIK  (Neurol.  Centralb.,  1897,  p.  521 ;  WEIGEBT  (op.  cit.,  supra, 
p.  5)  impregnates  as  Marchi,  and  afterwards  treats  with  permanganate  and 
oxalic  acid  as  in  the  method  of  PAL  (§  712). 

See  also  ROSSOLIMO  &  BUSCH,  Zeit.f.  wiss.  Mik.,  xiv,  1897,  p.  55. 

720.  AZOULAY'S  Osmic  Acid  Method  (Anat.  Anz.,  x,  1,  1894, 
p.  25). —  (A)  Sections  of  material  that  has  been  for  several 
months  in  liquid  of  Muller  are  put  for  five  to  fifteen  minutes 
into  solution  of  osmic  acid  of  1  :  500  or  1  :  1000  strength. 
Rinse  with  water  and  put  them  for  two  to  five  minutes  into 
a  5  or  10  per  cent,  solution  of  tannin,  warming  them  therein 
over  a  flame  till  vapours  are  given  off,  or  in  a  stove  at  50° 
to  55°  C.  Wash  for  five  minutes  in  water,  double-stain  if 
desired  with  carmine  or  eosin,  and  mount  in  balsam.  Thin 
sections  are  necessary  to  ensure  good  results.  If  they 
should  be  too  thick  it  will  be  necessary  after  staining  to 
differentiate  by  PAL'S  process,  or  by  eau  de  Javelle  diluted 


406  CHAPTER  XXXI. 

with  50  vols.  of  water.  (B)  Material  that  has  been  in  an 
osmic  mixture  (liquid  of  Flemming,  of  Marchi,  or  of  Grolgi)  .• 
Sections  as  before,  then  the  tannin  bath,  warming  for  three: 
to  ten  minutes,  and  the  rest  as  before. 

721.  HELLEE  and  GUMPERTZ  (Zeit.  f.  wiss.  Mik.,  xii,  1896, 
p.  385)  give  for  peripheral  nerves,  and  HELLEE  (op.  cit.,  xv, 
1899,  p.  495)  for  central  nervous  system,  the  following  :— 
The  material  is  hardened  in  liquid  of  Miiller.  Sections  (by 
the  celloidin  method  if  desired)  are  put  into  1  per  cent, 
osmic  acid  (twenty-four  hours  at  37°  C.)  for  peripheral 
nerves,  ten  minutes  (or  thirty  at  the  normal  temperature)  for 
central.  They  are  treated  with  pyrogallic  acid  (a  photo- 
graphic developer  will  do)  till  the  nerves  are  black,  then 
with  a  violet-coloured  solution  of  permanganate  of  potash 
till  the  sections  become  brown,  then  with  2  per  cent,  oxalic 
acid  till  they  become  yellow-green.  Wash  out  well  between 
each  operation;  the  preparations  are  then  permanent. 
Mount  in  glycerin  or  balsam. 

Similarly  KOBEBTSON  (Brit.  Mcd.  Journ.,  1897,  p.  651;  Journ.  Roy. 
Hie.  Soc.,  1897,  p.  175),  the  material  being  previously  mordanted  with 
Weigert's  chrome-alum-copper  fluid  for  neuroglia. 

722.  Silver    Nitrate.  —  VASTAEINI-CEESI   (Att.    Accad.  Med.    Chir. 
Napoli,  1,  1896)  hardens  in  forraol,  cuts  thick  sections,  washes  them  with 
40  per  cent,  alcohol,  puts  them  in  the  dark  into  1  per  cent,  solution  of 
nitrate  of  silver  in  alcohol  of  40  per  cent,  to  70  per  cent.,  then  washes 
thoroughly. 

723.  Gold  Chloride  for   Peripheral  Nerves  (FfiEY,  Arch.  Anat. 
Phys.,  Anat.  Abth.  Supp.,  1897,  p.  108.     See  Grundzilge,  LEE  &  MAYER, 
p.  421). 

724.  Polarisation. — Myelin  can  sometimes  be  detected  by  the  polari- 
scope  (see   AMBRONN  &   HELD,  Ber.   Math.  Phys.   Ges.   Wiss.  Leipzig, 
1895,  p.  37).      They  examined   teased  preparations  of  peripheral  nerves 
fresh  in  normal  salt  solution,  or  thick  sections  of  nerve  centres  cut  with 
the  freezing  microtome. 

See  also  GAD  &  HEYMANS,  Arch.  Anat.  Phys.,  Phys.  Abth.,  1890, 
p.  531. 


NEORO LOGICAL    METHODS.  407 


b.  Myelin-and-axis-cylinder   Stains. 

725.  PALADINO'S  Iodide  of  Palladium  Method  (Rendic  R. 
Accad.  Scienze  Fis.  e  Mat.,  Napoli,  iv,  1890,  p.  14,  and  1891 
[1892],  p.  227  ;  -Zeit.f.  wi*s.  Mik.,  vii,  2,  1890,  p.  237,  andix,- 
2,  1892,  p.  238;  Journ.  Roy.  Mic.  Soc.,  1890,  p.  817,  and 
1892,  p.  439). — Pieces  of  material  hardened  in  bichromate, 
chromic  acid,  or  corrosive  sublimate,  and  not  more  than  5  to 
8  mm.  in  thickness,  are  put  for  two  days  into  a  large  quan- 
tity (at  least  150  to  200  c.c.  for  each  piece)  of  0*1  per  cent, 
solution  of  chloride  of  palladium  (see  §  78) .  They  are  next 
put  for  twenty-four  hours  into  a  solution  of  iodide  of  potas- 
sium of  4  :  100  strength,  of  which  a  relatively  small  volume 
should  be  taken ;  otherwise  the  iodide  of  palladium,  which 
is  rapidly  formed  in  the  tissues,  may  be  again  extracted  by 
the  liquid  (small  pieces  of  tissue  should  not  remain  in  it  for 
more  than  one  or  two  hours) .  Dehydrate  ;  imbed,  if  neces- 
sary, in  paraffin  by  the  chloroform  method ;  mount  in 
balsam. 

Later  (Boll.  Accad*  Med.  Eon.  a,  xix,  1893,  p.  256  ;  Arch. 
ItaL  BioL,  xx,  1894,  p.  40)  he  first  dehydrates  the  pieces; 
then  puts  them  in  an  incubator  for  an  hour  into  absolute 
alcohol  and  benzol,  an  hour  in  pure  benzol,  and  finally 
twenty-four  hours  in  absolute  alcohol,  which  removes  the 
myelin.  They  are  then  put  for  a  week  into  chloride  of 
palladium  of  1  to  2  per  cent.,  one  to  two  days  into  4  per 
cent,  iodide  of  potassium,  and  are  lastly  passed  through 
alcohol  into  celloidin. 

726.  SAHLI  (Zeit.  f.  iviss.  Mik.,  1885,  p.  1)  stains  sections  of  tissue 
hardened  in  bichromate  to  the  degree  required  for  Weigert's  haematoxylin 
process  for  several  hours  in  concentrated  aqueous  solution  of  methylen 
blue,  rinses  with  water,  and  stains  for  five  minutes  in  saturated  aqueous 
solution  of  Saurefuchsin.  If  now  the  sections  be  rinsed  with  alcohol  and 
brought  into  a  liberal  quantity  of  water,  the  stain  becomes  differentiated, 
axis-cylinders  being  shown  coloured  red  and  the  myelin  sheaths  blue. 

The  same  author  (loc.  cit.,  p.  50)  also  gives  the  following: — Sections  of 
material  hardened  as  before  are  stained  for  a  few  minutes  or  hours  in  the 
following  liquid  : 

Water 40  parts. 

Saturated  aqueous  solution  of  methylen  blue  .     24      „ 
5  per  cent,  solution  of  borax  .         .         .         .     16      ,, 
(Mix,  let  stand  a  day,  and  filter.) 


408  CHAPTER  XXXI. 

The  sections  are  then  washed  either  in  water  or  alcohol  until  the  grey 
matter  can  be  clearly  distinguished  from  the  white,  are  cleared  with  cedar 
oil,  and  mounted  in  balsam.  Nerve  tubes  blue,  ganglion  cells  greenish, 
nuclei  of  neuroglia  blue. 

727.  Method  of  ADAMKIEWICS  (Sitzb.  k.  Akad.    Wiss.    Wien.  Math. 
Naturw.  KL,  1884,  p.  245 ;  Zeit.  f.  wiss.  Mik.,  1884,  p.  587).— Sections  (of 
spinal  cord  hardened  in  liquid  of  Muller  for  not  less  than  one  month  and 
not  more  than  three)  are  washed  first  with  water,  then  in  water  acidified 
with  a  little  nitric  acid,  and  stained  in  concentrated  solution  of  safranin. 
They  are  then  treated  with  alcohol  and  clove  oil  till  no  more  colour  comes 
away,  and  are  brought  back  again  into  water,  washed  in  water  acidified 
with  acetic  acid,  stained  in  methylen  blue,  and  cleared  as  before.     Myelin 
red,  nuclei  violet. 

NIKIFOBOW  (Zeit.  f.  wiss.  Mik.,  v,  3,  1888,  p.  338)  impregnates  with 
gold  chloride  or  other  metallic  salt  after  the  safranin  stain. 

Similarly  CIAGLLNSKI  (Zeit.  f.  wiss.  Mik.,  viii,  1,  1891,  p.  19)  and 
STEOEBE  (ibid.,  x,  3,  1893,  p.  336),  both  of  them  employing  safranin 
followed  by  anilin  blue. 

For  NISSL'S  Congo  red  method  see  Zeit.f.  wiss.  Mik.,  iii,  1886,  p.  398. 

728.  FINOTTI  (op.  cit.,  §  719)  stains  strongly  in  Delafield's  hseinatoxylin, 
then  for  a  few  seconds  in  concentrated  solution  of  picric  acid,  then  in  0'5 
per  cent.  Saurefuchsin,  and  treats  with  alkaline  alcohol  (caustic  potash). 

OHLMACHEE  (Journ.  Exper.  Med.,  ii,  1897,  p.  675)  stains  sections  on  the 
slide  for  one  minute  with  anilin-water  gentian,  §  272,  then  for  a  few 
seconds  in  a  solution  of  0*5  per  cent,  of  Saurefuchsin  in  saturated  solution 
of  picric  acid  diluted  with  one  volume  of  water,  washes  well  with  water, 
differentiates  with  alcohol  and  clove  oil,  and  mounts  in  balsam. 

729.  ARONSON  (Centralb.  med.  Wiss.,  1890,  p.  577)  stains  sections  of 
material  hardened  in  liquid  of  Erlicki  or  Muller  (these  must  be  mordanted 
with  acetate  of  copper)  for  twelve  to  twenty-four  hours  in  a  solution  of 
3  to  4  c.c.  of   Gallein  (Griibler  &  Co.)  in  100  c.c.  of  water  with  20  of 
alcohol  and  three  drops  of  concentrated  solution  of   carbonate  of    soda. 
They  are  then  differentiated  by  the  method  of  Weigert,  or  Pal,  or  with 
chloride  of  calcium,  §  717,  then  brought  into  concentrated  solution  of 
carbonate  of  soda  or  lithia  until  they  become  red,    and  are  mounted  in 
balsam  (clear  with  oil  of  origanum).     Nerve  fibres  red.     A  second  stain 
with  methylen  blue  may  follow  after  differentiating  with  permanganate. 


CHAPTER  XXXII. 

NEUROLOGICAL  METHODS,  AXIS-CYLINDER  AND  PROTOPLASM 
STAINS  (GOLGI  AND  OTHERS). 

730.  Introduction. — There  are  three  chief  methods  for  the 
study  of  axis-cylinders  and  protoplasmic  nerve-cell  processes, 
viz.  the  methylen-blue  staining  method,  the  sublimate  method 
of  GOLGI,  and  the  bichromate-of-silver  method  of  GOLGI.  The 
methylen-blue  method  having  been  given  in  Chap.  XVII,  it 
remains  to  group  together  here  some  other  subordinate  but 
useful  methods  that  are  also  stains  proper  •  after  which  will 
be  given  the  methods  of  GOLGI  and  some  other  impregnation 
methods. 

(c)    Stains  Proper. 

731.  Anilin  blue-black  has  been  much  recommended  by  SAN  KEY 
(Quart.  Journ.  Mic.  Sci.,  1876,  p.  69) ;  BE  VAN  LEWIS  (Human  Brain, 
p.  125  ;  VEJAS  (Arch.  f.  Psychiatric,  xvi,  p.  200)  ;  GIEKKE  (Zeit.  f.  wiss. 
Mik.,  1884,  p.  376) ;  MARTINOTTI  (ibid.,  p.  478) ;  JELGEESMA  (Zeit.f.  wiss. 
Mik.,  1886,  p.  39) ;  SCHMAUS  (Munch,  med.  Wochenschr.,  No.  8,  1891, 
p.  147;  Zeit.f.  wiss.  Mile.,  viii,  1891,  p.  230),  and  others.  I  have  not  been 
able  to  identify  the  colour  used  by  these  authors,  but  as  they 'concur  in 
saying  that  the  English  preparation  sold  under  that  name  alone  gives  good 
results,  I  conclude  that  it  must  have  been  the  anilin  black  of  Lightfoot. 
If  so,  it  is  no  longer  found  in  commerce,  and  should  not  be  quoted  as  a  his- 
tological  reagent  (see  §  313).  For  details  see  previous  editions. 

732.  MABTINOTTI  (loc.  cit.,  1884,  p.  478)  finds  that  picro- 
nigrosin  gives  very  good  results,  especially  for  pathological 
objects.  He  stains  for  two  or  three  hours  or  days  in  a 
saturated  solution  of  nigrosin  in  saturated  solution  of  picric 
acid  in  alcohol,  and  washes  out  in  a  mixture  of  1  part  of 
formic  acid  with  2  parts  of  alcohol  until  the  grey  matter 


410  CHAPTER    XXXli. 

appears  clearly  differentiated  from  the  white  to  the  naked 
eye. 

733.  KAISEE  (Zeit.f.  wiss.  Mik.,  vi,  4, 1889,  p.  471)  advises  naphthylamin 
brown  (Griibler).     Sections  of  spinal  cord  are  stained  for  a  few  hours  in  a 
solution  containing  1  part  of  naphthylainin  brown,  200  parts  of  water,  and 
100  parts  of  alcohol,  washed  with  alcohol,  cleared  with  origanum  oil,  and 
mounted. 

734.  KEHM  (Munch,  med.  Wochenschr.,   1892,  No.  13 ;  Zeit.  f.  wiss.. 
Mik.,  ix,  3,  1893,  p.  389)  gives  a  method  modified  from  NISSL.     Sections  of 
alcohol -hardened  material  are  stained  for  half  a  minute  to  a  minute  in  a 
hot  O'l  per  cent,  solution  of  methylen  blue,  washed  in  96  per  cent,  alcohol 
till  no  more  colour  comes  away,  cleared  with  origanum  oil,  and  mounted  in 
balsam  or  benzin-colophonium.     Nerve  cells,  dark  blue  ;  connective-tissue 
cells  lighter,  and  greenish.     For  further  details  see  previous  editions. 

735.  MONCKEBKRG  and  BETHE  (Arch.f.  mik.  Anat.,  liv,  J 899,. 
p.  135;  Zeit.f.  u'istf  Mik.,  xvi,  18^9,  p.  244)  recommend  (for 
peripheral  nerves  only)  the  following  : — Nerves  are  fixed  hi 
0*25  per  cent,  osmic  acid  for  twenty-four  hours  and  bleached 
with  bisulphite  of  sodium,  as  directed  §  38,  and  cut  in  paraffin. 
The  sections  are  stained  011  the  slide  for  ten  minutes  in  O'l 
per  cent,  solution  of  toluidin  blue,  warmed  to  50°  or  60°  C., 
washed  with  water  for  one  or  two  minutes,  then  treated  for 
a   few    seconds    or    minutes    with    1    per   cent,    solution    of 
molybdate  of  ammonium.      Water,  alcohol,  xylol,  balsam. 

Or  the  sections  are  first  mordanted  for  five  to  ten  minutes 
in  4  per  cent,  solution  of  molybdate  of  ammonium  warmed 
to  20°  or  30°  C.,  and  washed  with  water;  then  toluidin  blue 
solution  (of  0*05  to  O'l  per  cent.)  is  poured  on  to  the  slide, 
which  is  put  for  five  minutes  into  a  stove  at  50°  to  60°  C. 
Water,  alcohol,  xylol,  balsam. 

736.  WOLTER'S    Chloride    of   Vanadium    process    for    axis- 
cylinder  and  cell   staining  is  as  follows    (Zeit.  f.  iciss.  Mik.,. 
vii,  4,  1891,  p.  471)  : 

The  material  (either  central  or  peripheral  nervous  tissue) 
is  hardened  in  the  bichromate  liquid  of  KULTSCHITZKY,  §  59, 
followed  by  alcohol,  as  there  described.  Sections  are  mor- 
danted for  twenty-four  hours  in  a  mixture  of  2  parts  of  10 
per  cent,  solution  of  chloride  of  vanadium  and  3  parts  of 
3  per  cent,  solution  of  acetate  of  aluminium,  washed  for  ten 


NEUROLOGICAL    METHODS.  411 

minutes  in  water,  and  stained  for  twenty-four  hours  in  a 
solution  of  2  grammes  of  hasmatoxylin  (dissolved  in  a  little 
alcohol)  in  100  c.c.  of  2  per  cent,  acetic  acid.  They  are 
washed  out  until  they  are  of  a  light  blue-red  colour  in  80  per 
cent,  alcohol  acidulated  with  0'5  per  cent,  of  hydrochloric 
acid.  Remove  the  acid  thoroughly  by  washing  with  pure 
alcohol,  dehydrate,  clear  with  origanum  oil,  and  mount. 

A  sharp  axis-cylinder  stain,  myelin  being  coloured  only  if 
the  differentiation  in  the  acid  alcohol  is  insufficient. 

737.  SCARPATETTI  (Neurol.  Centralb.,  xvi,  1897,  p.  211 ;  Zeit.  f.  wiss. 
Mik.,  xiv,  1897,  p.  91)  obtains  an  axis-cylinder  stain  as  follows : — Sections 
of  material  hardened  in  5  to  10  per  cent,  formol,  followed  by  alcohol,  are 
stained  for  five  minutes  in  1  per  cent,  haeinatoxylin,  treated  for  five  minutes 
with  concentrated  solution  of  neutral  acetate  of  copper,  differentiated  with 
Weigert's  borax-ferricyanide,  then  treated  with  concentrated  solution  of 
carbonate  of  lithia,  washed,  and  mounted.  Myelin  is  not  stained. 

738.  Iron-Haematoxylin   sometimes   gives    an   axis-cylinder 
stain. 

739.  MALLORY'S  Phospho-molybdic-acid  Haematoxylin  has  been 
given,  §  259,  where  see  also  that  of  KENYON. 

For  the  extremely  complicated  modification  of  AUEBBACH,  see  Neurol. 
Centralb.,  xvi,  1897,  p.  439,  or  Zeit.  f.  wiss.  Mik.,  xiv,  1897,  p.  402. 

74.0.  FINOTTI  (Virchow's  Arch.,  cxliii,  1896,  p.  133;  Zeit.  f.  wiss.  Mik., 
xiii,  1896,  p.  236)  stains  in  haematoxylin,  washes  out  well,  counter-stains 
for  three  minutes  with  0'5  to  1  per  cent,  solution  of  Saurefuchsin,  and 
differentiates  in  75  per  cent,  alcohol  containing  a  very  little  caustic  potash. 
See  also  §  704. 

741.  ALT  (Munch,  med.  Wochenschr.,  1892,  No.  4;  Zeit.  f.  wiss.  Mik., 
ix,  1,  1892,  p.  81)  stains  for  a  couple  of  hours  in  solution  of  Congo  in  abso- 
lute alcohol,  and  washes  out  with  pure  alcohol.  The  results  are  said  to  be 
specially  adapted  to  the  study  of  peripheral  axis-cylinders. 

742.  APATHY'S  methods.      See  §  707. 


(d)    Impregnations. 

743.  The  Methods  of  GOLGI.      There   are  two  methods    of. 
GOLGI,  viz.  the  Corrosive  Sublimate  Method  and  the  Bichromate 


412  CHAPTER   XXXII. 

and  Nitrate  of  Silver  Method.  The  corrosive  sublimate  method 
will  be  given  later  on. 

The  bichromate  and  nitrate  of  silver  method  has  been 
worked  out  by  Golgi  in  three  forms.  These  are  known  as 
the  slow  process,  the  rapid  process,  and  the  mixed  process.* 

The  rapid  process  is  the  one  that  is  the  most  in  use  at  the 
present  time  for  researches  into  the  distribution  and  relations 
of  axis-cylinders  and  protoplasmic  processes;  it  may  be  taken 
to  be  the  classical  method  of  inquiry  into  the  finer  relations 
of  the  neurons  in  hardened  tissue. 

General  characters  of  the  impregnation. — The  preparations 
have  not  in  the  least  the  appearance  of  stains,  and  are  even 
very  different  in  aspect  from  the  impregnations  obtained  on 
fresh  tissue  by  the  ordinary  methods  of  impregnating  with 
nitrate  of  silver  or  chloride  of  gold.  The  impregnation  is  a 
partial  one,  by  which  is  meant  that  of  all  the  elements, 
whether  nervous  or  not,  that  are  present  in  a  preparation, 
only  a  limited  number  are  coloured.  That  is  the  peculiar 
quality — not  by  any  means  the  defect,  but  rather  the  advan- 
tage— of  the  method.  For  if  all  the  elements  present  were 
coloured  equally,  with  the  great  intensity  with  which  they 
take  the  colour  in  this  method,  you  would  not  be  able  to  see 
the  wood  for  the  trees,  in  fact  you  would  hardly  be  able  to 
distinguish  any  detail  at  all  in  the  preparations.  But  Golgi' s 
method  selects  from  among  the  elements  present  a  small 
number  which  it  stains  with  a  great  intensity  and  very  com- 
pletely ;  that  is  to  say,  throughout  a  great  length,  so  that 
they  are  both  very  clearly  separated  from  those  elements  that 
have  remained  uncoloured,  such  as  supporting  cells  and  the 
like,  and  also  can  be  followed  out  for  a  great  distance. 

Axis- cylinders  are  only  impregnated  so  long  as  they  are 
not  medullated.  In  the  adult  the  method  stains  nerve  cells 
and  their  processes,  so  far  as  these  are  not  myelinated ;  but 
if  it  be  wished  to  impregnate  the  nerve  fibres  of  the  cerebro- 
spinal  axis,  the  method  must  be  applied  to  embryos  or  new- 

*  In  a  recent  text-book,  the  Leitfaden  of  RAWITZ,  the  sublimate  method 
is  called  "  the  slow  method  of  GOLGI,"  and  the  bichromate  and  silver  nitrate 
method  is  given  under  the  form  of  the  slow  process,  and  called  "the  rapid 
method  of  GOLGI."  That  is  a  very  "  nice  derangement  of  epitaphs  "  indeed. 
RAWITZ  further  attributes  the  rapid  method  to  RAMON  Y  CAJAL,  which  is 
equally  erroneous.  Similar  confusions  are  made  by  MEECIEE  in  his  Coupes 
du  Systeme  Nerveux  Central. 


NEDROLOGICAL   METHODS.  413 

born  animals  at  a  time  when  the  fibres  have  not  become 
surrounded  by  their  sheath  of  myelin. 

There  is  no  other  method  which  will  allow  cell-processes 
to  be  followed  out  for  such  great  distances.  But  the  method 
does  not  demonstrate  at  the  same  time  the  histological  detail 
of  other  tissues  that  may  be  present  in  the  preparations,  and 
all  cytological  detail  is  lost.  It  it  par  excellence  a  special 
method. 

Nervous  tissue  is  not  the  only  thing  that  is  impregnated 
in  these  preparations ;  neuroglia,  connective  tissue,  fibrils,  etc., 
are  impregnated,  and  the  method  has  been  applied  with 
success  to  the  study  of  such  things  as  bile-capillaries,  gland- 
ducts,  and  the  like.  Both  on  account  of  this  character,  and 
on  account  of  the  capriciousness  with  which  the  impregnation 
takes  hold  of  only  certain  elements  of  the  preparations,  care 
must  be  exercised  in  the  interpretation  of  the  images  ob- 
tained. As  with  gold  impregnations,  the  very  best  prepara- 
tions give  images  that  are  only  worthy  of  credence  as  to  what 
they  show,  and  furnish  absolutely  no  evidence  whatever  as  to 
the  non-existence  of  anything  that  they  do  not  show ;  for  you 
can  never  be  sure  that  the  imbibition  of  the  salt  has  not 
capriciously  failed,  or  its  reduction  capriciously  stopped,  at 
any  point.  And  a  further  source  of  error  is  found  in  the 
fact  that  the  method  frequently  gives  precipitation-forms  of 
the  silver  salt  that  simulate  dendrites  and  other  structures 
(see  FRIEDLAENDER  in  Zeit.  f.  wiss.  Mik.,  xii,  2,  1895,  p.  168, 
and  the  plate  in  the  following  number).  A  correspondent 
writes  me  that  he  has  "  Golgified  a  potato,  and  obtained 
beautiful  nerve-fibres/'  and  FKIEDLAENDER'S  paper  describes 
similar  results  obtained  with  white  of  egg,  etc.  And  other 
workers  have  made  similar  observations.  Clearly,  then, 
much  caution  is  necessary  in  the  interpretation  of  the 
images. 

The  method  has  been  applied  with  success  to  the  tissues 
of  Invertebrates — Insects,  Liimbricus,  Tubifex,  Helix,  Limax, 
Distomum,  Astacus,  etc. 

The  details  of  the  method  have  been  considerably  modified 
at  the  hands  of  various  workers,  the  most  important  modifica- 
tion being  that  of  the  "  double  "  or  "  intensified  "  impregna- 
tion of  RAMON  Y  CAJAL. 

The  method  has  been  described  at  length  by  GOLGI  in  the 


414  CHAPTER    XXXII. 

Archives  Italiennes  de  Biologie,  t.  iv,  1883,  p.  32,  et  seq.,  and 
vii,  1886,  p.  15,  et  seq.  The  following  account  is  from  the 
latter  paper.  The  earlier  form  of  the  method  should  not  be 
followed. 

744.  GOLGI'S  Bichromate  and  Nitrate  of  Silver  Method,  SLOW 
Process  (loc.  cit.,  p.  17). —  (a)  The  hardening. — This  must  be 
done  in  a  bichromate  solution.  Either  pure  bichromate  of 
potash  may  be  employed,  or  liquid  of  Miiller  (the  reaction 
can  be  obtained  with  liquid  of  Erlicki,  but  it  is  not  to  be 
recommended).  The  normal  practice  is  to  take  bichromate 
of  potash,  beginning  with  a  strength  of  2  per  cent.,  and 
changing  this  frequently  for  fresh  solutions  of  gradually 
increased  strength,  2£,  3,  4,  and  5  per  cent.  The  tissue  to 
be  operated  on  should  be  as  fresh  as  possible  ;  though  satis- 
factory results  may  be  obtained  from  material  taken  twenty- 
four  to  forty-eight  hours  after  death/'  It  should  be  in  pieces 
of  not  more  than  1  c.cm.  or  1  \  c.cm.  in  size. 

The  most  difficult  point  of  the  method  consists  in  hitting 
off  the  exact  degree  of  hardening  in  the  bichromate  that 
should  be  allowed  before  passing  to  the  next  stage  of  the 
process,  the  silver-bath.  In  summer  good  results  may  be 
obtained  after  fifteen  to  twenty  days,  and  the  material  may 
continue  in  a  favourable  state  for  impregnation  up  to  thirty, 
forty,  or  fifty  days.  In  cold  weather  good  results  can 
seldom  be  obtained  under  a  month ;  when  obtained,  the 
material  may  continue  to  give  good  results  up  to  two,  three, 
and  even  four  months  of  hardening.  The  only  way  to  make 
sure  is  to  pass  trial  portions  of  the  tissue  at  intervals  into 
the  silver-bath,  in  summer  frequently,  in  winter  every  eight 
or  ten  days,  and  observe  whether  the  reaction  is  obtained. 

Good  results  are  obtained  by  injecting  the  organs  with 
the  hardening  fluid  (2*5  per  cent,  bichromate).  See  §  681. 

Stoving  at  a  temperature  of  20°  to  25°  C.  is  useful  for 
-abridging  the  hardening,  but  there  is  risk  of  over-hardening ; 
and  GOLGI  thinks  the  results  are  never  quite*  so  delicate  as 
after  hardening  in  the  cold. 

(b)  Impregnation. — As  soon  as  the  pieces  of  tissue  have 
attained  the  proper  degree  of  hardening,  they  are  brought 
into  a  bath  of  nitrate  of  silver.  The  usual  strength  of  this 
bath  is  0'75  per  cent.,  but  0'50  per  cent,  may  be  taken  for 


NEUROLOGICAL    METHODS.  415 

material  that  has  not  been  quite  enough  hardened,  and  solu- 
tions of  1  per  cent,  may  be  used  for  material  that  has  been 
slightly  over-hardeued. 

A  relatively  large  quantity  of  solution  should  be  taken 
for  the  bath. 

The  moment  the  pieces  of  tissue  are  put  into  the  silver- 
bath,  an  abundant  yellow  precipitate  of  chromate  of  silver 
is  formed.  This  of  course  weakens  the  bath  pro  tanto.  It 
is  therefore  well,  before  putting  the  pieces  into  the  final 
silver-bath,  to  first  wash  them  well  in  a  weaker  silver  solu- 
tion, until  on  being  put  into  a  fresh  quantity  of  it  no  farther 
precipitate  is  formed.  Used  solutions  will  do  for  this  pur- 
pose. The  final  silver-bath  in  general  needs  no  further 
attention,  unless  it  be  that  sometimes,  in  the  case  of  tissues 
that  have  taken  up  a  great  deal  of  bichromate  of  potash, 
the  solution  may  after  six  to  ten  hours  become  somewhat 
yellow,  in  which  case  it  should  be  changed  for  fresh. 

It  is  not  necessary  to  keep  the  preparations  in  the  dark 
during  the  impregnation-bath  ;  in  winter  it  is  well  to  keep 
them  in  a  warm  place. 

The  time  necessary  for  impregnation  by  the  silver  is  from 
twenty-four  to  forty-eight  hours.  The  normal  time  is  from 
twenty- four  to  thirty  hours,  forty-eight  being  quite  excep- 
tional. By  this  is  meant  that  the  reaction  is  not  obtained 
in  less  time,  but  tissues  may  remain  in  the  bath  without  hurt 
for  days,  weeks,  or  months. 

(c)  Preservation. — As  soon  as  a  trial  has  shown  that  a 
sufficiently  satisfactory  impregnation  has  been  obtained,  the 
pieces  are  brought  into  alcohol.  The  alcohol  is  changed 
two  or  three  times,  or  even  more,  until  it  remains  trans- 
parent even  after  the  preparations  have  been  two  or  three 
days  in  it ;  for  in  view  of  good  preservation  it  is  necessary 
that  the  excess  of  nitrate  of  silver  should  be  washed  out  from 
them  thoroughly. 

Sections  are  now  made.  They  are  to  be  washed  very 
thoroughly  in  three  or  four  changes  of  absolute  alcohol. 
They  are  then  cleared,  first  in  creosote,  in  which  they  should 
remain  only  a  few  minutes,  then  in  oil  of  turpentine,  in 
which  they  should  remain  for  ten  to  fifteen  minutes  (they 
may  remain  there  for  days  without  hurt).  They  are  then 
mounted  in  damar  (rather  than  in  balsam),  and  without  a 


416  CHAPTER   XXXII. 

cover.  Preparations  mounted  under  covers  in  the  usual  way 
always  go  bad  sooner  or  later,  whilst  those  that  are  mounted 
without  a  cover  keep  very  well,  especially  if  they  be  kept  in  the 
dark.  GOLGI  states  that  he  has  a  large  number  that  have 
kept  without  change  for  nine  years. 

The  order  in  which  the  elements  of  tissues  impregnate  is 
— first,  axis-cylinders,  then  ganglion  cells,  and  lastly  neu- 
roglia  cells. 

745.  GOLGI' s    Bichromate   and   Nitrate   of    Silver    Method, 
RAPID  Process  (op.  cit.,  p.  33).      Small  pieces  of  very  fresh 
tissue  are  thrown  into  the  following  mixture  : 

Bichromate  solution  of  2  to  2' 5  per  cent. 

strength         .  .  ,  .  .8  parts. 

Osmic  acid  of  1  per  cent,  strength          .      2     „ 

The  hardeniog  being  much  more  rapid  than  with  the 
slow  process,  the  tissues  will  begin  to  be  in  a  fit  state  for 
taking  the  silver  impregnation  from  the  second  or  third 
day  ;  in  the  next  following  days  they  will  be  in  a  still  more 
favourable  state,  but  the  favourable  moment  does  not  last 
long  ;  the  faculty  of  impregnation  soon  declines,  and  is  gene- 
rally quite  lost  by  the  tenth  or  twelfth  day. 

The  silver  impregnation  is  conducted  exactly  as  in  the 
slow  process,  and  sections  are  prepared  and  mounted  in  the 
same  manner. 

There  is  this  difference,  that  the  impregnated  material 
cannot  be  preserved  for  any  length  of  time  in  alcohol,  but 
must  not  remain  for  more  than  two  days  in  it.  But  it  may 
be  kept  in  the  silver  solution  until  wanted  for  sectioning*. 

This  process  has  the  advantage  of  great  rapidity,  and  of 
sureness  and  delicacy  of  result,  and  is  the  one  that  has 
found  the  most  favour  with  other  workers.  But  for 
methodical  study  of  any  given  part  of  the  nervous  system, 
G-OLGI  himself  prefers  the  following  : 

746.  GOLGI'S    Bichromate  and    Nitrate    of   Silver    Method, 
MIXED    Process    (op.    cit.,  p.  34). — Fresh  pieces  of  tissue 
are  put   for  periods  varying  from    two    to    twenty-five    or 
thirty  days  into  the  usual  bichromate  solution.      Every  two 
or  three  or  four  days  some  of  them  are  passed  on  into  the 
osmio-bichromate   mixture   of  the    rapid  process,   hardened 


NEUROLOGICAL    METHODS.  417 

therein  for  from  three  or  four  to  eight  or  ten  days,  and 
finally  impregnated  with  silver,  and  subsequently  treated 
exactly  as  in  the  rapid  process. 

The  reasons  for  which  GOLGI  prefers  this  process  are — the 
certainty  of  obtaining  samples  of  the  reaction  in  many  stages 
of  intensity,  if  a  sufficient  number  of  pieces  of  tissue  have 
been  operated  on  ;  the  advantage  of  having  at  one's  dis- 
position a  notable  time — some  twenty-five  days — during 
which  the  tissues  are  in  a  fit  state  for  taking  the  silver,  and 
the  possibility  of  greatly  hastening  the  process  whenever 
desired  by  simply  bringing  the  pieces  over  at  once  into  the 
osmic  mixture ;  lastly,  a  still  greater  delicacy  of  result, 
especially  remarkable  in  the  demonstration  of  the  "  func- 
tional "  or  nervous  process  of  nerve  cells. 

747.  Critique  of  GOLGI'S  Method. — The  above-described 
methods  have  been  found  extremely  valuable  in  the  most 
various  departments  of  nervous  anatomy.  They  have  given 
brilliant  results  in  the  study  of  peripheral  nerves  and  their 
origins  or  terminations,  and  in  the  study  of  the  relations  of 
fibres  and  cells  in  the  central  nervous  system.  It  has  been 
found  at  the  same  time  that  they  have  the  defect  of  con- 
siderable uncertainty  in  the  production  of  the  desired 
reaction,  and  in  the  preservation  of  the  stain.  These  defects 
have  given  rise  to  a  most  elaborate  discussion,  which  un- 
happily has  not  as  yet  led  to  very  satisfactory  results. 

GOLGI'S  method  is  apparently  (but  this  is  by  no  means 
certain)  based  on  the  formation  in  the  tissues  of  a  precipitate 
of  bichromate  of  silver  which  is  brown  by  reflected  light, 
but  appears  black  by  transmitted  light.  The  problem  is  to 
preserve  this  precipitate  in  the  tissues  free  from  chemical 
or  molecular  change.  And  the  problem  is  not  an  easy  one ; 
without  special  precautions  the  preparations  will  not  resist 
the  processes  necessary  for  imbedding,  will  not  always  resist 
those  necessary  for  merely  mounting  in  balsam,  and  even 
then  may  easily  "  go  bad  "  after  they  have  been  mounted  for 
a  short  time. 

A  critical  review  of  the  Golgi  method  by  WEIGEET  may  be  found  in 
Ergebnisse  der  Anatomic,  v,  1895  (1896),  p.  7.  He  thinks  the  precipitate 
certainly  consists  of  a  silver  chromate,  but  that  we  cannot  say  which. 

The  method  has  also  been  critically  studied  by  HILL  (Brain,  part  73, 

27 


418  CHAPTER   XXXII. 

1896,  p.  1).  He  thinks  the  stain  depends  on  the  formation  of  a  "  reduced 
salt  (subsalt)  of  silver,"  not  of  a  silver  chromate,  and  that  the  reduction 
takes  place  not  in  the  nervous  fibrils,  but  in  the  liquid  or  semi-liquid 
"neuroplasm"  with  which  they  are  bathed.  He  finds  the  impregnated 
material  will  stand  imbedding  in  celloidin  for  many  days.  For  impregna- 
tion he  recommends  instead  of  silver  nitrate  a  f  per  cent,  solution  of  silver 
nitrite  with  (H  per  cent,  of  formic  acid  added.  Other  details  loc.  cit. 

AZOULAY  (Comptes  Rend.  Soc.  Biol.  [10],  i,  1894,  p.  839)  has  followed 
the  process  under  the  microscope,  and  holds  that  it  is  due  to  a  crystallisa- 
tion of  chromate  of  silver  in  the  tissues. 


Modifications  concerning  the  Impregnation  of  the  Tissues. 

748.  RAMON   Y   CAJAL,  who  has   done  a  great  deal  of  im- 
portant work  by  GOLGI'S  method,  has  always  used  the  rapid 
process.      For  the  times  and  strengths  used  by  him  in   his 
researches  on  the  cerebral  cortex  of  mammals   see  his  paper 
in  La  Cellule,  vii,  1891,  p.   125,  or  Zeit.  f.   wiss.  Mile.,  ix,  2, 
1892,  p.  239;  also  Journ.  Roy.  Mic.  Soc.,  1892,  p.  154.      He 
found  it  useful  to  adopt  SEHRWALD'S  gelatin  process   (§  757) 
for  avoidance  of  peripheral  precipitates.      He  prefers  not  to 
adopt     GREP PIN'S    treatment    with    hydrobromic    acid,    nor 
OBREGIA'S  treatment  with  gold  chloride,  finding  that,  although 
they  serve  to  render  the  preparations  permanent,  they  obscure 
the  finer  relations  of  fibres. 

For  embryos  of  the  fowl  he  employs  the  same  process  ; 
see  his  paper  in  Anat.  Anz.,  v,  1890,  85,  or  Zeit.  f.  wiss.  Mile., 
vii,  2,  1890,  p.  285. 

749.  RAMON  Y  CAJAI/S  Double-Impregnation  Process. — In  a 
paper  on  the  structure  and  relations  of  the  sympathetic  gan- 
glia  (which  I  have  not  seen,  and  quote  from   Zeit.  f.  wiss. 
Mik.,  loc.  cit.)  RAMON  Y  CAJAL  describes  a  process  of  ' '  intensi- 
fied "  or  "  double  impregnation."  After  hardening  for  three 
days    (embryos  of  fowl)   in  the  osmium-bichromate  mixture, 
the  preparations  are  put  for  thirty- six  hours  into  nitrate  of 
silver  solution  (0*5  to  0'75  per  cent.).      They  are  then  brought 
back   into  the  same  osmium-bichromate  mixture,  or  into  a 
weaker  one  containing  only  two  parts  of  osmic  acid  solution 
to  20  of  the  bichromate.      After  treatment  with  this  they  are 
washed  quickly  with  distilled  water,  and  put  for  a  second 


NEUROLOGICAL    METHODS.  419 

time  into  the  silver  solution  for  thirty-six  to  forty-eight 
hours.  It  is  important  to  hit  off  the  proper  duration  of  the 
first  impregnation  in  the  bichromate.  If  it  has  been  too  long 
(four  days)  or  too  short  (one  day),  the  second  impregnation 
will  not  succeed.  In  this  case  a  third  impregnation  must  be 
resorted  to,  the  objects  being  again  treated  with  the  weak 
osmium-bichromate  mixture,  and  afterwards  again  with  the 
silver  solution. 

This  modification  of  the  original  process  is,  perhaps,  the 
most  important  that  has  hitherto  been  made. 

750.  KALLIUS  (Anat.  Hefte,  x,  1894,  p.  527;  Zeit.  f.  wiss.  Mile.,  xi,  2, 

1894,  p.  154)  states  that  he  has  often  found  it  advantageous  to  employ 
bichromate  of  ammonia  or  of  soda  instead  of  the  bichromate  of  potash,  and 
to  perform   all   the    reactions  in   the   dark.      Preparations   made   by  the 
ammonia  or  soda  salt  rarely  require  a  double  impregnation. 

751.  BOEHM,  and  afterwards  OPPEL  (Anat.  Anz.,  v,  1890,  p.  143,  and 
vi,  1891,  p.  165;  Zeit.  f.  wiss.  Mik.,  vii,  2,  1890,  p.  222,  and  viii,  2,  1891, 
p.  224),  have  modified  the  hardening  part  of  the  process  by  taking  instead 
of  bichromate  of  potash  (slow  process)  the  one  an  0'5  per  cent,  solution  of 
chromic  acid  (forty-eight  hours),  the  other  a  solution  of  neutral  chromate 
of  potash  of  from  0'5  per  cent,  to  as  much  as  10  per  cent.     This  is  for 
liver. 

BERKLEY  (Anat.  Anz.,  1893,  p.  772)  fixes  pieces  of  liver  for  fifteen  to 
thirty  minutes  in  warm  half-saturated  solution  of  picric  acid,  and  hardens 
for  forty-eight  hours  in  a  stove  in  the  dark  in  a  "sunned"  (§  336)  mixture 
of  sixteen  parts  2  per  cent,  osmic  acid  and  100  parts  saturated  solution  of 
bichromate. 

752.  Formaldehyde  Mixtures. — STRONG  (Anat.  Anz.,  x,  15, 

1895,  p.  494)   states  that  formaldehyde  can  with  advantage 
be   substituted   for   the   osmic   acid   in   the  osmio-bichromic 
mixture  of  GOLGI'S  rapid  process.      He  adds  from  2 '5  to  5  per 
cent,  of  "formaline"  to  the  3'5  to  5  per  cent,  bichromate 
solution. 

The  advantage  is  stated  to  be  that  the  stage  of  hardening 
favourable  for  impregnation  lasts  longer ;  in  other  words,  the 
formaldehyde  bichromate  does  not  over-harden. 

DURIG  (ibid.,  p.  659)  obtained  the  best  results  by  means 
of  3  per  cent,  bichromate  solutions  containing  4  to  6  per  cent, 
of  formaldehyde,  hardening  therein  for  three  days,  and  then 
performing  double  impregnation  by  RAMON  Y  CAJAI/S  process. 


420  CHAPTER  XXXII. 

FISH    (Proc.  Amer.  Mic.  Soc.,  xvii,  1895,  p.  319)   has  also 
obtained  good  results  with  the  following  mixtures  : 

Formalin    ......        2  c.c. 

3  per  cent,  bichromate  .  .  .   100    „ 

leaving  the  tissues  three  days  in  this  liquid  and  three  days  in 
the  silver  nitrate  (f  per  cent.). 

Or,  with  advantage  : 

Liquid  of  Miiller          .  .  ^  .  100  c.c., 

10  per  cent,  formalin  .  .  .  2    „ 

1  per  cent,  osmic  acid  .  .  .  1    „ 

The  formalin  and  bichromate  mixtures  should  be  kept  in 
the  dark.  It  is  well  only  to  make  them  up  at  the  instant  of 
using  them. 

KOPSCH  (Anat.  Anz.,  xi,  1896,  p.  727)  states  that  he  has 
obtained  good  impregnations  with  a  mixture  of  4  parts  of 
3*5  per  cent,  bichromate  solution,  and  one  of  commercial 
formaldehyde  solution.  He  considers  the  results  more  certain 
than  with  the  osmic  acid  mixture. 

G-EEOTA  (Intern.  Monatsschr.  Anat.,  xiii,  1896,  p.  108 ;  Zeit. 
f.  wiss.  Mik.,  xiii,  1896,  p.  314)  first  hardens  (brain)  for  a 
week  or  two  in  5  to  10  per  cent,  formol  solution,  then  puts 
small  pieces  for  three  to  five  days  into  4  per  cent,  bichromate, 
then  into  the  silver. 

SCHEEIBEE  (Anat.  Anz.,  xiv,  1898,  p.  275)  obtained  good 
results  (on  appendages  of  Crustacea  which  were  impervious 
to  the  osmic  mixture)  with  mixtures  of  five  parts  2*5  per  cent, 
bichromate  to  one  of  4  per  cent,  formaldehyde,  or  one  part 
2'5  per  cent,  bichromate  to  two  of  5  per  cent,  formaldehyde, 
the  specimens  remaining  for  one  day  in  the  first,  for  two  days 
in  the  second. 

BOLTON  (Lancet,  1898,  p.  218;  Journ.Roy.  Mic.  Soc.,  1898, 
p.  244)  hardens  brain  for  five  weeks  or  more  in  formalin, 
then  puts  pieces  for  a  few  hours  to  five  days  into  1  per  cent, 
bichromate  of  ammonia. 

Similarly  BAEI  (Zeit.  f.  wiss.  Mik.,  xvi,  2,  1899,  p.  243), 
using  2  per  cent,  bichromate  of  potash. 

VAN  G-EHOCHTEN  (in  litt.)  has  tried  the  substitution  of 
formaldehyde  for  the  osmic  acid  in  the  G-OLGI  process,  and 
has  not  obtained  good  results. 


NEUROLOGICAL   METHODS.  421 

753.  Acetic  Aldehyde. — VASSAL  K   and   DONAGGIO    (Monitor e 
Zool.,  Ital.,  vi,  1895,  p.  82)  harden  pieces  of  at  most  1  cm. 
in  thickness  for  fifteen  to  twenty  days  in  a  mixture  of  five 
parts  of  aldehyde  with   100  of  3  to  4  per  cent,  bichromate, 
changing   the   fluid   after   a   few  days,   as    soon   as   it   has 
become  dark.     The  rest  as  Golgi. 

754.  Hardening  by  Injection. — This  was  recommended  by 
GOLGI,  see  §§  744  and  681.      He  found,  however,  that  the 
bichromate    caused    such   an    energetic   contraction    of    the 
arterioles    that    the    injection   did    not    penetrate    into   the 
capillaries.      TOOTH  got  over  this  by  adding  morphia  to  the 
injection. 

HILL  (op.  cit.,  §  747)  gets  over  it  by  injecting  'through 
the  aorta,  whilst  the  heart  is  still  beating,  a  solution  of 
bichromate  containing  1  per  cent,  of  lactic  acid. 

For  nitrite  of  amyl  as  a  vaso-dilator,  see  §  476. 

755.  Reviving   Over-hardened    Tissues. — Tissues   that   have 
been  too  long  (three  to  four  weeks)  in  the  osmium-bichromate 
mixture  will  no  longer  take   on  the  silver  impregnation,  as 
has    been    explained    above.      They    can,    however,    be    re- 
vivified   and  made  to  impregnate  in  the  following  manner, 
due     to     GOLGI,     and     published     by    SACEBDOTTI     (Intern. 
Monatsschr.,  xi,  1894,  6,  p.  326 ;   Zeit.  f.  wiss.  Mik.,  xi,  3, 
1894,  p.  389).      They  are  washed  in  a  half-saturated  solution 
of  acetate  of  copper  until  they  no  longer  give  a  precipitate, 
and  are  then  put  back  again  for  five  or  six  days  into  the 
osmium-bichromate    mixture.      Sections  of  the  impregnated 
material  give  remarkably  fine  images,  and  will  bear  mount- 
ing in  thickened  oil  of  cedar  under  a  cover. 

756.  Modifications  of  the  Silver  Impregnation. — KOLOSSOW  (Arch, 
f.  mik.  Anat.,  xlix,  1897,  p.  592)  (after  hardening  in  osmio-bichromate 
solution)  impregnates  for  two  or  three  days  in  a  2  to  a  3  per  cent,  solution 
of  silver  nitrate  in  %  to  £  per  cent,  osmic  acid  solution,  and  states  he  thus 
obtains  a  more  complete  impregnation. 

Similarly  JUSCHTSCHENKO,  see  Zeit.f.  wiss.  Mik.,  xiv,  1897,  p.  82. 

BEBKELEY  (Johns  Hopkins  Hosp.  Rep.,  vi,  1897,  p.  1 ;  Journ.  Roy.  Mic. 
Soc.,  1898,  p.  242)  impregnates,  after  hardening  in  the  osmio-bichromate, 
in  a  freshly  prepared  solution  of  two  drops  of  10  per  cent,  phospho- 
molybdic  acid  to  60  c.c.  of  1  per  cent,  silver  nitrate,  which  in  winter 
should  be  kept  at  a  temperature  of  about  25°  C. 


422  CHAPTER    XXXII. 

757.  SEHRWALD'S  Gelatin  Process  (Zeit.  f.  iviss.  Mik.,  vi,  4, 
1889,  p.  456). 

GOLGI'S  process  frequently  gives  rise  to  the  formation  at  the 
surface  of  the  preparations  of  voluminous  precipitates  that 
are  destructive  of  the  clearness  of  the  images.  SEHRWALD 
finds  that  this  evil  can  be  avoided  by  putting  the  tissues 
into  gelatin  solution  before  bringing  them  into  the  silver- 
bath.  A  10  per  cent,  solution  of  gelatin  in  water  may  be 
made.  The  tissues  are  imbedded  in  this,  in  a  paper  im- 
bedding box,  with  the  aid  of  a  little  heat  (the  gelatin  melting 
at  a  sufficiently  low  temperature),  and  are  brought  therein 
into  the  silver-bath.  After  the  silvering  the  gelatin  is 
removed  by  warm  water  saturated  with  chromate  of  silver. 
MARTINOTTI  wraps  the  tissues  simply  in  blotting-paper,  but 
this  does  not  appear  to  be  efficacious. 


Modifications  concerning  the  Preservation  of  the  Preparations. 

758.  Cutting  and  Mounting. — Many  most  elaborate  methods 
have  been  proposed  with  the  object  of  fixing  the  stain  so 
that  the  preparations  may  bear  imbedding  in  paraffin  and 
the  sections  bear  mounting  under  a  cover.  None  of  them 
have  met  with  much  favour. 

SALA  (Zeit.  f.  wiss.  Zool.,  lii,  1,  1891,  p.  18;  Zeit.  f.  wiss. 
Mik.,  viii,  3,  p.  389),  in  a  paper  written  in  Golgi's  labora- 
tory, finds  Greppin's  hydrobromic  acid  variation  (§  759)  not 
merely  useless,  but  hurtful.  And  he  thinks  that  SEHRWALD' s 
process  (infra)  for  imbedding  .the  material  in  paraffin  with 
the  object  of  getting  very  thin  sections  is  a  mistake.  The 
chief  quality  of  GOLGI'S  process  is  that  it  admits  of  the 
following  of  nerve-cell  processes  for  a  very  great  distance. 
Evidently  this  cannot  be  done  with  very  thin  sections.  It 
is  better  simply  to  wash  the  preparations  taken  from  the 
silver-bath  with  water,  fix  them  to  a  cork  with  gum,  put 
the  whole  into  alcohol  for  a  few  hours  to  harden  the  gum, 
and  cut  with  a  microtome  without  imbedding. 

An  elaborate  discussion  (for  which  see  previous  editions) 
between  SEHRWALD  (Zeit.  f.  wiss.  Mik.,  vi,  1890,  p.  443), 
SAMASSA  (ibid.,  vii,  1890,  p.  26),  and  FICK  (ibid.,  viii,  1891, 
p.  168)  furnishes  the  net  practical  result  that  watery  fluids 


NEUROLOGICAL   METHODS.  423 

should  be  avoided  as  much  as  possible  during  the  after- 
treatment,  and  that  sections  should  either  be  mounted  with- 
out a  cover,  or  on  a  cover  raised  free  of  contact  with  the 
slide  by  means  of  wax  feet  or  the  like ;  or  that  the  balsam 
of  the  mount  should  be  rendered  perfectly  anhydrous  by 
careful  heating  on  the  slide,  with  the  section  in  it,  until  it 
immediately  sets  hard  on  cooling,  before  the  cover  is 
applied. 

This  last  method  is  also  recommended  by  HUBER  (Anat. 
Am.,  vii,  1892,  p.  587;  Journ.  Roy.  Hie.  Soc.,  1892,  p.  707; 
Zeit.  f.  wiss.  Mik.,  ix,  4,  1893,  p.  479) . 

The  majority  of  workers  seem  to  abide  by  this  result,  and 
to  consider  that  the  fixation  methods  shortly  given  in  the 
next  four  sections  are,  to  say  the  least,  superfluous. 

759.  GEEPPIN'S  Process  (Arch.  f.  Anat.  u.  Entw.,  Anat.  Abth.,  1889, 
Supp.,  p.  55 ;   Zeit.  f.  wiss.  Mik.,  vii,  1,  1890,  p.  66). — After  silvering, 
sections  are  made  with  a  freezing  microtome  and   treated  for  thirty  to 
forty  seconds  with  10  per  cent,  solution  of  hydrobromic  acid,  and  may  then 
be  well  washed  in  several  changes  of  water  and  mounted  under  a  cover  in 
the  usual  way.     Further  details  in  previous  editions. 

760.  OBBEGIA'S  Process  (Virchow's  Archiv,  cxxii,  1890,  p.  387;  Zeit. 
f.  wiss.  Mik.,  viii,  1,  1891,  p.  97 ;  Journ.  Boy.  Mic.  Soc.,  1891,  pp.  536, 

830;  Amer.  Mon.  Micr.  Journ.,  1891,  p.  210). — Sections  of  silvered 
material  are  made,  either  without  imbedding,  or  after  imbedding  either  in 
paraffin  or  celloidin,  care  being  taken  in  either  case  not  to  use  alcohol  of  a 
lower  grade  than  94  or  95  per  cent.  They  are  brought  from  absolute 
alcohol  into  a  mixture  of  eight  to  ten  drops  of  1  per  cent,  solution  of  gold 
chloride  with  10  c.c.  of  absolute  alcohol,  which  should  be  prepared  half  an 
hour  beforehand  and  exposed  to  diffused  light  until  the  sections  are  placed 
in  it,  when  it  should  be  put  into  the  dark.  After  fifteen  to  thirty  minutes 
therein,  according  to  their  thickness,  the  sections  are  quickly  washed  in  50 
per  cent,  alcohol,  then  in  water,  then  treated  for  five  or  ten  minutes  with 
10  per  cent,  solution  of  hyposulphite  of  soda.  They  are  lastly  washed  well 
with  water,  and  may  then  be  mounted  at  once  in  balsam  under  a  cover,  or 
if  desired  may  be  previously  stained  with  carmine  or  haernatoxylin,  or  Pal's 
modification  of  Weigert's  process,  or  the  like. 

761.  KALLIUS  (Anat.  Hefte,  ii,  1892,  p.  269  ;  Zeit.  f.  wiss.  Mik.,  ix,  4, 
1893,  p.  477)  has  worked  out  the  following  process.     Take  20  c.c.  commercial 
hydroquiuone  developing  solution  and  230  c.c.  distilled  water  (the  hydro- 
quinone  solution  may  be  made  up  with  5   grins,  hydroquinone,  40  grms. 
sodium  sulphite,  75  grms.  carbonate  of  potassium,  and  250  grms.  distilled 
water).     At  the  instant  of  using,  further  dilute  the  solution  with  one  third 


424  CHAPTER   XXXII. 

to  one  half  its  volume  of  absolute  alcohol,  and  put  the  sections  into  it  for 
several  minutes  ;  they  become  dark  grey  to  black.  As  soon  as  reduction  is 
complete  the  sections  are  put  for  ten  to  fifteen  minutes  into  70  per  cent, 
alcohol,  then  brought  for  five  minutes  into  solution  of  hyposulphite  of 
soda  (about  10  parts  to  50  of  water),  and  thence  into  a  large  quantity  of 
distilled  water,  where  they  should  remain  for  twenty -four  hours  or  more. 
Lastly,  dehydrate  in  the  usual  way  and  mount  under  a  cover.  After- 
staining  with  carmine,  etc.,  may  be  employed. 

762.  ZIMMEEMANN'S  Process  (Arch.  mik.  Anat.,  lii.  1898,  p.  552  ;  Zeit. 
f.  wiss.Mik.,xv,  1898,  p.  216). — Sections  are  brought,  after  silvering,  from 
alcohol  into  a  mixture  of  1  part  physiological  salt  solution  and  2  parts 
96  per  cent,  alcohol.  They  must  be  kept  in  motion  therein  for  ten  to 
fifteen  minutes,  after  which  they  are  brought  into  alcohol  of  75  to  96  per 
cent.,  in  a  bright  light,  until  they  have  become  dark. 


The  Sublimate  Method. 

763.  GrOLGi's  Bichromate  and  Sublimate  Method  (Archivio  per 
le  Scienze  Mediche,  1878,  p.  3;  Archives  Italiennes  de  Biologie, 
iv,  1883,  p.  32  ;  vii,  1886,  p.  35).— This  method  consists  of  two 
processes  :  1,  hardening  in  bichromate  ;  2,  treatment  with 
bichloride  of  mercury. 

For  hardening,  use  either  a  solution  progressively  raised 
in  concentration  from  1  per  cent,  to  2|  per  cent.,  or  Miiller's 
solution.  It  is  beat  to  take  small  pieces  of  tissue  (not  more 
than  1  to  2  c.c.),  large  quantities  of  liquid,  and  change  the 
latter  frequently,  so  as  to  have  it  always  clear.  But  the  re- 
action can  be  obtained  with  much  larger  pieces,  even  entire 
hemispheres.  In  this  case  the  brain  should  at  first  be  treated 
by  repeated  injections  of  the  liquid,  so  as  to  ensure  as  rapid 
a  permeation  of  the  interior  as  possible.  Fifteen  to  twenty 
days'  immersion  will  suffice,  or  even  six  to  eight,  but  twenty 
to  thirty  should  be  preferred,  and  an  immersion  of  several 
months  is  not  injurious. 

The  tissues  when  hardened  are  passed  direct  from  the 
bichromate  into  0'5  per  cent,  solution  of  bichloride  of  mer- 
cury. An  immersion  of  eight  to  ten  days  therein  is  necessary 
in  order  to  obtain  a  complete  reaction  through  the  whole 
thickness  of  the  tissues  (or  for  entire  hemispheres  two  months 
or  more).  During  the  bath  the  bichromate  will  diffuse  out 
from  the  tissues  into  the  bichloride,  which  must  at  first  be 


NEUROLOGICAL    METHODS.  425 

changed  every  day,  and  later  on  as  often  as  it  becomes 
yellow.  At  the  end  of  the  reaction  the  preparations  will  be 
found  decolourised,  and  offering  the  aspect  of  fresh  tissue. 
They  may  be  left  in  the  bichloride  for  any  time. 

In  Rendiconti  R.  1st.  Lombardo  di  Sci.  Milano,  2,  xxiv,  1891,  pp.  594r 
656  (see  Zeit.  f.  wiss.  Mik.,  viii,  3,  1891,  p.  388),  GOLGI  says  that  for  the 
study  of  the  diffuse  nervous  reticulum  of  the  central  nervous  system  the 
best  results  are  obtained  by  keeping  the  preparations  in  1  per  cent,  sub- 
limate for  a  very  long  time,  two  years  being  not  too  much  in  some  cases. 

The  reaction  may  be  said  to  have  begun  by  the  time  the 
tissues  are  nearly  decolourised.  From  that  time  onwards 
sections  may  be  made  day  by  day  and  examined,  and  those 
which  it  is  desired  to  preserve  may  be  mounted. 

Before  mounting,  the  sections  that  have  been  cut  must  be 
repeatedly  washed  with  water  (if  it  be  wished  to  mount  them 
permanently),  otherwise  they  will  be  spoilt  by  the  formation 
of  a  black  precipitate.  (In  the  last  place  quoted  GOLGI  says 
that  after  washing  they  may  be  toned  by  putting  them  for  a 
few  minutes  into  a  photographic  fixing-and-toning  bath,  after 
which  it  is  well  to  wash  them  again,  and  stain  them  with 
some  acid  carmine  solution.)  Mount  in  balsam  or  glycerin  ; 
the  latter  seems  the  better  preservative  medium. 

The  result  of  this  process  is  not  a  true  stain,  but  an 
"  apparently  black  reaction/7  the  tissues  appearing  black  by 
transmitted  light,  u'hite\)y  reflected  light.  GOLGI  thinks  that 
there  is  formed  in  the  tissue  elements  a  precipitate  of  some 
substance  that  renders  them  opaque.  The  elements  acted  on 
are — (1)  The  ganglion  cells,  with  all  their  processes  and  rami- 
fications of  the  processes.  These  are  made  more  evident  than 
by  any  other  process  except  the  bichromate  and  silver-nitrate 
process.  (2)  J\ruclei,  which  is  not  the  case  with  the  silver 
process.  (3)  Connective-tissue  corpuscles  in  their  charac- 
teristic radiate  form.  But  the  reaction  in  this  case  is  far  less 
precise  and  complete  than  that  obtained  by  the  silver  process. 
(4)  The  blood-vessels,  and  particularly  their  muscular  fibre 
cells. 

The  method  gives  good  results  only  with  the  cortex  of  the 
cerebral  convolutions,  hardly  any  results  at  all  with  the 
spinal  cord,  and  very  scanty  results  with  the  cerebellum. 
And,  on  the  whole,  the  method  shows  nothing  more  than 


426  CHAPTER  XXXII. 

can  be  demonstrated  by  the  silver-nitrate  method,  but  it  is 
superior  to  it  in  that  the  reaction  can  always  be  obtained 
with  perfect  certainty  in  a  certain  time ;  that  the  prepara- 
tions can  be  perfectly  preserved  by  the  usual  methods ;  and 
that  large  pieces  of  tissue  can  be  impregnated. 

See  also  FLATATT,  in  Arch.f.  mile.  Anat.,  xlv,  1895,  p.  158 ;  Zeit.f.  wiss. 
Mik.,  xii,  2,  1895,  p.  257. 

The  method  is  recommended  by  BLOCHMANN  (Biol.  Centralb.,  xv,  1895, 
p.  14 ;  Zeit.  f.  wiss.  Mik.,  xii,  2,  1895,  p.  226)  for  the  nervous  system  of 
Cestodes. 


Modifications  of  Golgi's  Bichromate  and  Sublimate  Method. 

764.  TAL  (Gazz.  degli  Ospitali,  1886,  No.  68)  finds  that  if  sections  made 
by  this  process  be  treated  with  solution  of  sodium  sulphide,  a  much  darker 
stain  is  obtained.  Sections  may  then  advantageously  be  double-stained  with 
Magdala  red. 

Golgi's  method  may  be  combined  with  Weigert's  nerve  stain  (see  PAL, 
Wien.  med.  Jahrb.,  1886  ;  Zeit.f.  wiss.  Mik.,  v,  1,  1887,  p.  93). 

765.  Cox  (Arch.  f.  mik.  Anat.,  xxxvii,  1891,  p.  16;  Journ. 
Roy.  Mic.  Soc.,  1891,  p.  420)  finds  the  sublimate  and  bichro- 
mate may  be  used  together,  and  give  a  uniform  impregnation. 
He  used  a  fluid  consisting  of  20  parts  5  per  cent,  bichromate, 
20  parts  5  per  cent,  sublimate,  16  parts  5  per  cent,  simple 
chr ornate  of  potash,  and  30  to  40  parts  of  water.  (The 
simple  chromate  should  be  added  to  the  other  ingredients 
only  after  diluting  with  the  water.)  The  mixture  should  be 
as  little  acid  as  possible.  The  duration  of  the  impregnation 
is  from  two  to  three  months.  There  is  considerable  difficulty 
in  preserving  sections,  which  must  be  made  with  a  freezing 
microtome,  alcohol  being  avoided,  treated  for  an  hour  or  two 
with  5  per  cent,  solution  of  sodium  carbonate,  and  mounted 
without  a  cover. 

Dr.  A.  SANDEES,  on  the  other  hand,  writes  me  (June,  1898)  that  the  stain 
keeps  very  well  if  the  sublimate  be  well  removed  by  washing  in  many 
changes  of  alcohol,  and  the  tissues  passed  through  alcohol  and  ether  into 
celloidin,  and  the  sections  mounted  in  chloroform- balsam  under  a  cover.  I 
think  the  statement  is  correct  so  far  as  regards  the  preservation  of  the 
stain  ;  but  the  preparations  quickly  develop  opaque  granules  that  are  very 
unsightly. 


NEUROLOGICAL   METHODS.  427 

760.  MAGINI'S  Zinc  Chloride  Process  (see  Boll.  Accad.  Med.  di  Roma, 
1886;  Zeit.f.  wiss.Mik.,  1888,  p.  87,  or  previous  editions}. 

767.  FLECHSIG'S  modifications,  see  Arch.  f.  Anat.  u.  Phys.,  Physiol. 
Abth.,  1889,  p.  537 ;  Zeit.f.  wise.  Mik.,  vii,  1,  1890,  p.  71. 

768.  MONTI'S  Copper  Process,  see  Atti  d.  R.  Accad.  dei  Lincei  Roma, 
Rendic.,  \,  1889,  1  sem.,  p.  705  ;  Zeit.f.  wiss.  Mik.,  vii,  1,  1890,  p.  72. 

769.  GEKLACH'S  Bichromate   and   Gold  Process   has  been  given, 
§356. 


Other  Methods. 

770.  ZIEHEN'S  Gold  and  Sublimate  Method  (Neurol.  Centralb., 
x,  1891,  No.  3,  p.  65;  Zeit.  f.  wiss.  Mik.,  viii,  3,    1891,  p. 
385). — Small   pieces   of    fresh  material  are   thrown  into  a 
large  quantity  of  a  mixture  of  1  per  cent,  sublimate  solution 
and    1   per   cent,  chloride   of  gold   solution  in  equal  parts. 
They  remain  therein  for  at   least  three  weeks,  preferably  for 
several  months  (up   to   five),  by  which  time  they  will  have 
become  of  a  metallic  red-brown  colour.      They  are  gummed 
on  cork  and  sectioned  without  imbedding.      The  sections  are 
treated  either  with  Lugoi's  solution  (§  88)  diluted  with  four 
volumes   of  water,   or  with    dilute    tincture   of   iodine,  until 
duly   differentiated,    then    washed   and   mounted  in  balsam. 
The  result  is  a  bluish-grey  impregnation ;   both    medullated 
arid  non-medullated  nerve-fibres  are  stained,  also   nerve  and 
glia  cells  and  their  processes. 

771.  APATHY'S  Gold  Method  has  been  given  (§  358). 

772.  For  UPSON'S  exceedingly  complicated  Gold  and  Iron 
and  Vanadium  Methods  see  ME«C[ER,  in  Zeit.f.  wiss.  Mik.,  vii, 
4,    1891,    p.  474;    or  in  his   "Coupes  du  Systeme  Nerveux 
Central,  p.  234,  or  previous  editions. 

773.  KBOHNTHAI/S  Lead  Sulphide  Impregnation  (Neurol.  Cen- 
tralb., xviii,   1899,   No.    5;   Zeit.f.  wi*x.  Mik.,  xvi,  1899,  p. 
235)  consists  in  treating   tissues   first   with  formate  of  lead 
and  then  with  hydric  sulphide.      The  formate  is  prepared  by 


428  CHAPTER  XXXII. 

dropping  formic  acid  slowly  into  solution  of  acetate  of  lead. 
White  crystals  of  formate  of  lead  are  abundantly  formed  ; 
the  mother  liquor  is  filtered  off,  and  the  crystals  are  dis- 
solved to  saturation  in  water.  The  solution  is  mixed  with 
an  equal  volume  of  10  per  cent,  formol  solution ;  pieces  of 
brain  or  spinal  cord  are  put  into  the  mixture  for  five  days, 
and  are  then  brought  direct  into  a  mixture  of  equal  parts  of 
10  per  cent,  formol  solution  and  hydric  sulphide  solution. 
After  five  days  therein  they  are  passed  through  successive 
alcohols,  imbedded  in  celloidin,  cut,  and  the  sections  mounted 
in  xylol-balsam  under  a  cover.  They  seem  to  be  quite  per- 
manent. Nerve  cells  as  well  as  nerve  fibres  are  impregnated, 
and  it  is  the  elements  themselves  that  are  impregnated,  and 
not  lacuna?  around  them,  as  appears  to  be  the  case  with  the 
Grolgi  impregnation.  The  impregnation  is  a  very  complete 
one. 

CORNING  (Anat.  Anz.,  xvii,  1900,  p.  108)  hardens  the 
tissues  with  10  per  cent,  formol  before  bringing  them  into 
the  formol -formate  mixture,  and  so  obtains  better  results. 
He  obtains  his  formate  of  lead  direct  from  MERCK  (Plwn.bum 
formicicum).  He  thinks  the  celloidin  imbedding  injurious, 
and  prefers  to  cut  without  imbedding.  He  prefers  to  clear 
sections  with  clove  oil.  The  method  appears  to  him  par- 
ticularly useful  for  the  medulla  oblongata,  with  which  the 
Grolgi  method  does  not  succeed.  It  appears  likely  to  be 
very  useful  in  pathological  researches,  and  also  for  the 
naked -eye  differentiation  of  white  and  grey  matter,  as  well 
as  for  the  other  purposes  for  which  the  Golgi  method  is 
generally  employed.  Other  details  loc.  cit. 

774.  ROBERTSON^  Platinum  Impregnation.     See  Journ.  Roy.  Mic. 
Soc.,  1899,  p.  665. 

775.  WEIGERT'S  Specific  Neuroglia  Stain  (WEIGEKT'S  Beitr. 
zur  Kenntnisx  der  normalen  men*  ch  lichen  Neuroglia,  Frank - 
furt-a-M.,  1895  ;  quoted  from  Neurol.  Centralb.,  1895,  xxiii, 
p.  1146). — Pieces  of  tissue  of  not  more  than  half  a  centi- 
metre in  thickness  are  put  for  at  least  four  days  into  "  10  per 
cent,  solution  of  formol."  They  are  then  mordanted  for  four 
or  five  days  in  an  incubating  stove  (or  for  at  least  eight  days 
at  the  temperature  of  the  laboratory)  in  a  solution  containing 


NEUROLOGICAL   METHODS.  429 

5  per  cent,  of  neutral  acetate  of  copper,  5  per  cent,  of 
acetic  acid,  and  2J  per  cent,  of  chrome  alum,  in  water. 
(Add  the  alum  to  the  water,  raise  to  boiling  point,  and  add 
the  acetic  acid  and  the  acetate,  powdered.) 

After  the  mordanting  the  tissues  are  washed  with  water, 
dehydrated,  imbedded  in  celloidin,  and  sectioned.  The  sec- 
tions are  treated  for  ten  minutes  with  a  one  third  per  cent, 
solution  of  permanganate  of  potash,  and  well  washed  in 
water.  They  are  then  treated  for  two  to  four  hours  with  a 
solution  of  "  Chromogen."  "  Chromogen  "  is  a  naphthalin 
compound  prepared  by  the  Hoechst  dye  manufactory.  The 
solution  of  "  Chromogen  "  to  be  used  is  prepared  as  follows  : 
5  per  cent,  of  "  Chromogen  "  and  5  per  cent,  of  formic 
acid  are  dissolved  in  water,  and  the  solution  carefully  filtered. 
To  90  c.c.  of  the  filtrate  are  added  10  c.c.  of  10  per  cent, 
solution  of  sodium  sulphite. 

After  this  bath,  the  sections  are  put  till  next  day  into  a 
saturated  (5  per  cent.)  solution  of  Chromogen. 

They  are  next  carefully  washed  and  submitted  to  the 
stain.  The  stain  is  a  modification  of  WEIQKKT'S  fibrin  stain. 
Instead  of  saturated  aqueous  solution  of  methyl  violet,  you 
take  a  warm-saturated  solution  in  70  per  cent,  or  80  per 
cent,  alcohol,  decant  it  after  cooling,  and  add  to  it  5  per 
cent,  of  aqueous  solution  of  oxalic  acid ;  and  instead  of 
treating  with  pure  anilin,  you  take  a  mixture  of  equal  parts 
of  anilin  and  xylol.  This  is  afterwards  removed  from  the 
sections  by  means  of  pure  xylol,  and  they  are  mounted  in 
balsam. 

MALLORY  (Journ.  Exper.  Med.,  1897,  p.  532)  finds  that 
tissues  can  be  mordanted  for  the  stain  by  means  of  a  chrome 
salt.  He  fixes  them  for  four  days  in  10  percent,  solution  of 
formalin,  then  for  four  to  eight  in  saturated  solution  of  picric 
acid  (or  for  the  same  time  in  a  mixture  of  the  two),  then 
mordants  for  four  to  six  days  at  37°  C.  in  5  per  cent,  solu- 
tion of  bichromate  of  ammonia,  makes  sections  (celloidin)  and 
stains  them  in  WEIGERT'S  fibrin  stain. 

See  also  the  modification  of  Weigert's  stain  by  STORCH,  Virchow's  Arch., 
clvii,  1899,  p.  127 ;  Zeit.f.  wiss.  Mik.,  xvi,  4,  1900,  p.  475. 

776.  Saurerubin  for  Neuroglia. — KULTSCHITZKY  (Anat.  Anz., 
viii,  1893,  p.  357)  stains  paraffin  sections,  either  for  five  to 


430  CHAPTER   XXXII. 

ten  seconds  with  a  mixture  of  1  grm.  Saurerubin  (Rubin  S.), 
400  c.c.  2  per  cent,  acetic  acid,  and  400  c.c.  saturated  solu- 
tion of  picric  acid,  or  for  half  an  hour  in  a  mixture  of  3  to  5 
c.c.  of  the  above  stain  with  100  c.c.  of  96  per  cent,  alcohol, 
and  washes  out  well  with  alcohol. 

POPOW  (Zeit.  f.  wiss.  Mik.,  xiii,  1896,  p.  358)  makes  up 
the  stain  by  adding  a  few  drops  of  tincture  of  iodine  to  a 
1  per  cent,  solution  of  Saurerubin  (Rubin  S.). 

BUECKHAKDT  (La  Cellule,  xii,  1897,  p.  364)  also  finds  that 
Saurerubin  gives  a  better  stain  than  Saurefuchsin,  and 
recommends  I  part  of  saturated  aqueous  solution  thereof  to 
9  parts  of  picric  acid  solution  of  1  :  300  strength.  Nuclei 
may  be  previously  stained  with  methyl  violet. 


777.  Methylen  Blue  for  Central  Nervous  System  (SEMI  MEYEE, 
Arch.  f.  mik.  Anat.,  xlvi,  1895,  p.  282,  and  xlvii,  1896,  p.  734).— MEYER 
has  obtained  good  results  (for  the  central  nervous  system,  not  for  the  peri- 
pheral) by  means  of  subcutaneous  injection.  Large  quantities  of  solution 
must  be  injected.  A  young  rat  will  require  at  least  5  c.c.  of  1  per  cent, 
solution;  a  rabbit  of  a  few  weeks  about  40  c.c.  But  it  is  better  to  employ 
stronger  solutions,  5  to  6  per  cent.  The  total  dose  should  be  given  in 
several  portions,  at  intervals  of  one  to  several  hours.  It  is  not  necessary  to 
wait  till  death  by  intoxication  has  taken  place,  and  after  a  suitable  interval 
the  subject  maybe  killed.  It  is  not  necessary  to  expose  the  organs  to  the  air 
for  the  sake  of  "  oxydising  "  the  stain.  They  should  be  thrown  direct  into 
the  bath  of  BETHE,  §  329.  The  liquid  ought  to  be  well  cooled  before  use. 
The  preparations  should  remain  in  it  till  the  next  day. 

KAMON  Y  CAJAL  (Rev.  trim.  Micr.,  Madrid,  i,  1896,  p.  123  ;  Zeit.  f.  wiss. 
Mik.,  xiv,  1897,  p.  92)  stains  by  "  propagation  "  or  "  diffusion."  The  brain  is 
exposed  (rabbit)  and  the  cortex  is  divided  into  slices  of  a  couple  of  millimetres 
thickness  by  means  of  a  razor.  The  slices  are  then  covered  on  both  sides 
either  with  finely  powdered  methylen  blue,  or  with  a  saturated  solution  of 
the  same,  the  slices  are  replaced  in  their  natural  positions,  the  brain -case  is 
replaced  for  half  an  hour,  after  which  the  slices  are  removed  and  fixed  for 
a  couple  of  hours  with  Bethe's  ammonium  molybdate,  washed,  hardened 
for  three  or  four  hours  in  a  mixture  of  1  part  1  per  cent,  platinum  chloride, 
40  parts  formol,  and  60  parts  water,  further  treated  with  platinum  chloride 
of  1  in  300,  passed  through  alcohol,  and  if  small  enough  imbedded  in 
paraffin. 

778.  GIACOMINI'S  "Dry"  Process  for  Preserving  Brains  (Arch. 
per  le  Scienze  Mediche,  1878,  p.  11).  See  previous  editions. 


NEUROLOGICAL    METHODS.  431 


Retina.* 

779.  Fixation  and  Hardening. — For  section  cutting,  the 
retina  of  small  eyes  is  best  prepared  by  fixing  the  entire  un- 
opened bulb  with  osmium  vapour.  According  to  KANVIER 
(Traite,  p.  954)  you  may  fix  the  eye  of  a  triton  (without 
having  previously  opened  the  bulb)  by  exposing  it  for  ten 
minutes  to  vapour  of  osmium.  The  sclerotic  being  very 
thin  in  this  animal,  such  a  duration  of  exposure  is  generally 
sufficient.  Then  divide  it  by  an  equatorial  incision,  and  put 
the  posterior  pole  for  a  few  hours  into  one  third  alcohol. 

Somewhat  larger  eyes,  such  as  those  of  the  sheep  and 
calf,  may  be  fixed  in  solutions  without  being  opened.  But 
it  is  generally  the  better  practice  to  make  an  equatorial 
incision,  and  free  the  posterior  hemisphere  before  putting  it 
into  the  liquid. 

The  older  practice  was  to  use  strong  solutions  of  pure 
osmic  acid ;  but  most  of  the  best  recent  work  has  been  done 
with  chromic  mixtures. 

Dr.  Lindsay  Johnson  tells  me  that  he  now  gets  the  best 
results  by  fixing  the  globe  over  the  steam  of  a  1  per  cent, 
osmic  acid  solution  raised  to  the  temperature  at  which  vapour 
is  seen  to  be  given  off  (but  not  to  boiling-point),  for  five 
minutes  in  the  case  of  human  adults,  or  for  one  to  three 
minutes  in  the  case  of  human  infants,  all  monkeys  and  small 
mammals,  as  in  them  the  sclerotics  are  very  thin.  As  soon 
as  the  sclerotic  is  felt  to  be  firm  to  the  touch,  it  should  be 
opened  by  a  small  nick  with  a  razor  just  behind  the  ciliary 
body ;  or  if  the  eye  be  that  of  an  adult,  the  cornea  and  lens 
may  be  removed.  The  eye  is  then  put  for  twelve  hours  into 
the  mixture,  §  49 ;  it  is  then  washed  in  running  water,  and 
suspended  in  a  large  volume  of  2 '5  per  cent,  bichromate  of 
potash  for  two  days,  then  passed  gradually  through  successive 
alcohols,  beginning  with  20  per  cent.,  and  ending  with 
absolute,  taking  five  days  from  first  to  last. 

Other  hardening  liquids,  however,  also  give  good  results, 
provided  that  the  fixation  by  the  osmic  acid  has  been 

*  Besides  the  sources  quoted  in  the  text,  see  on  this  subject  SELIGMANN, 
Die  mikroskopischen  Untersuchungsmethoden  des  Auges,  Berlin,  S.  Karger 
(Kavlstrasse  13),  1899,  pp.  iv,  248. 


432  CHAPTER   XXXII. 

properly  performed :  amongst  them  liquid  of  Flemming,  and 
that  of  Miiller.  Formaldehyde  mixtures  he  does  not  recom- 
mend. 

LEBEE  (Munch,  med.  Wochenschr.,  xli,  30,  1894;  cf.  Zeit.f.  wiss.  Mik., 
xii,  1895,  p.  256)  confirms  Hermann's  observation  concerning  eyes  (see 
p.  76).  He  advises  a  4  per  cent,  solution  (forraol  1,  water  10).  After  a 
few  days'  hardening  in  this,  the  eyes  may  l>e  cut  through,  it  is  said, 
without  derangement  of  the  parts.  The  retina  lies  flat,  and  is  at  least  as 
well  preserved  as  with  solution  of  Miiller.  The  eyes  may  be  passed  with- 
out  hurt  direct  into  successive  alcohols  ;  the  vitreous  will  shrink  a  little, 
but  less  rather  than  more  than  after  solution  of  Miiller.  1  doubt  the 
correct  cytological  preservation  of  the  elements  by  this  process. 

See  also  HIPPEL  (Arch.  f.  Ophthalm.,  xlv,  1898,  p.  286 ;  Zeit.  f.  iviss. 
Mik.,  xvi,  1,  1899,  p.  79),  who  finds  that  formol  fixes  the  lens  badly,  the 
retina  well,  so  far  at  least  as  the  absence  of  folds  from  shrinkage  is  con- 
cerned. 


780.  Staining. — RAMON  Y  CAJAL  employs  the  rapid  method 
of  G-OLGI,  double-impregnation  process,  §  749. 

KUHNT  (Jen.  Zeit.f.  Natwrw.,  Bd.  xxiv,  H.  1,  1889,  p.  177  ; 
Zeit.  f.  wiss.  Mik.,  vii,  1,  1890,  p.  65)  employs  Pal's  modifi- 
cation of  Weigert's  haematoxylm  process. 

DOGIEL  employs  the  methylen-blue  method,  §§  326,  328, 
330. 

SCHAKKKE  (Sitzb.  fc.  Akad.  Wiss.  Wien,  xcix,  1890,  3,  p.  110; 
Zeit.  /.  wiss.  Mik.,  viii,  2,  1891,  227)  recommends  mordant- 
ing sections  in  1  per  cent,  chromic  acid  for  some  hours, 
washing  for  a  short  time  only  with  water,  staining  for  twenty 
hours  in  Kultschitzky's  acetic  acid  haematoxylin  (§  714),  and 
differentiating  for  twelve  hours  in  Weigert's  ferri cyanide 
solution  (§  710). 

KRAUSE  (loc.  cit.,  §  782)  treats  fresh  retina  with  perchloride  of  iron  or  of 
vanadium  in  1  per  cent,  solution,  and  then  with  a  2  per  cent,  solution  of 
tannic  or  pyrogallic  acid.  These  reagents  only  stain  the  granule  layers 
and  the  nuclei  of  the  ganglion  cells.  The  elements  of  the  other  layers  may 
then  be  stained  with  Saurefuchsin  or  some  other  anilin. 

LENNOX  (Arch.  f.  Ophthalm.,  xxxii,  1 ;  Zeit.  f.  wiss.  Mik.,  iii,  3,  1886, 
p.  408 ;  and  Journ.  Roy.  Mic.  Soc.,  1887,  p.  339)  has  applied  Weigert's 
haematoxylin  method  to  the  retina. 

CUCCATI  stains  with  concentrated  aqueous  solution  of  Saurefuchsin,  and 
mounts  in  balsam. 

See  also  BEENHEIMEE,  Sb.  k.  Akad.  Wiss.  Wien,  1884;  or  Journ.  Roy. 
Mic.  Soc.,  1886,  p.  167  ;  and  RAMON  Y  CAJAL,  Rev.  trim,  de  Hist.  Norm,  y 


NEUROLOGICAL    METHODS.  483 

Path.,  i,  1888,  p.  1 ;  Anat.  Am.,  1889,  p.  Ill;  Zeit.  f.  wias.  Mik.t  v,  3, 
1888,  p.  373,  and  vi,  2,  1889,  p.  204. 

COLUCCI  (Zeit.f.  wiss.  Mile.,  xii,  1,  1895,  p.  87)  recommends  Paladino's 
iodide  of  palladium  impregnation,  §  725. 

For  Bleaching  see  §§  575 — 583. 


781.  Sections. — Some  workers  recommend  celloidin ;  but  I 
consider  paraffin  preferable.      Sections  may  be  mounted  in 
dammar  or  (FLEMMING)  in  glycerin. 

782.  Dissociation   Methods. — For    maceration    preparations 
you  may  use  weak  solutions  (0'2  to  0*5  per  cent.)  of  osmic 
acid  for  fixation,  and  then  macerate  in  0*02  per  cent,  chromic 
acid  (M.  SCHULTZE),  or  in  iodised  serum  (M.  SCHULTZE),  or  in 
dilute  alcohol  (LANDOLT),  or  in  Miiller's  solution,  or  (RANVIER, 
Traite,  p.  957)  in  pure  water,  for  two  or  three   days.      THIN 
(Joum.  of  Anat.,   1879,  p.  139)  obtained  very  good  results 
by  fixing  for  thirty-six   to  forty-eight   hours   in   one   third 
alcohol,  or  in  25  per  cent,  alcohol,  and  then  staining  and 
teasing. 

SCHIEFFERDECKER  macerates  fresh  retina  for  several  days  in 
the  methyl  mixture,  §  545. 

KRAUSE  (Intern.  Monatssch.  f.  Anat.  u.  Hist.,  1884,  p.  225  ; 
Zeit.f.  wiss.  Mik.,  1885,  pp.  140,  396)  recommends  treatment 
for  several  days  with  10  per  cent,  chloral  hydrate  solution  ; 
the  rods  and  cones  are  well  preserved. 


Inner  Ear. 

783.  SCBWALBK  (Beitr.  z.  Phys.,  1887 ;  Zeit.  f.  wiss.  Mik., 
iv,  1,  1887,  p.  90;  Journ.  Roy.  Mic.  Soc.,  1887,  p.  840).— Fix 
(cochlea  of  guinea-pig)  for  eight  to  ten  hours  in  "  Flemming," 
wash  in  water,  decalcify  (twenty-four  hours  is  enough)  in  1 
per  cent,  hydrochloric  acid,  wash  the  acid  out,  dehydrate,  and 
imbed  in  paraffin.  See  also  FERRBRI,  §  573  (phloroglucin) . 

PRENANT  (Intern.  Monatsschr.  f.  Anat.  u.  Physiol.,  ix,  1, 
p.  6;  Zeit.  f.  wiss.  Mik.,  ix,  3,  1893,  p.  379).— For  sections, 
open  the  cochlea  in  solution  of  Flemming  or  of  Hermann, 
and  fix  therein  for  four  to  five  hours.  Avoid  decalcification 
as  far  as  possible,  as  it  is  inimical  to  good  preservation  of 

28 


434  CHAPTER    XXXIT. 

elements;  but  if  necessary  take  1  per  cent,  palladium  chloride. 
Make  paraffin  sections. 

Isolation  preparations  of  the  stria  vascularis  may  be  made 
by  putting  a  cochlea  for  a  day  into  1  per  cent,  solution  of 
osmic  acid,  then  for  four  to  five  days  into  0*1  per  cent,  solu- 
tion; the  stria  may  then  be  got  away  whole. 

784.  Other  Methods. — WALDEYEE,  Strieker's  Handb.,  p.  958  (decalci- 
fication  either  in  O'OOl  per  cent,  palladium  chloride  containing  10  per  cent, 
of  HC1,  or  in  chromic  acid  of  0*25  to  1  per  cent.). 

UBBAN  PEITCHAED  (Journ.  Roy.  Mic.  Soc.,  1876,  p.  211). — Decalcifica- 
tion  in  1  percent,  nitric  acid. 

LAVDOWSKY  (Arch.  f.  mik.  Anat.,  1876,  p.  497). — Fresh  tissues  (from 
the  cochlea)  are  treated  with  1  per  cent,  solution  of  silver  nitrate,  then 
washed  for  ten  minutes  in  water  containing  a  few  drops  of  0'5  or  1  per  cent, 
osmic  acid  solution,  and  mounted  in  glycerin. 

MAX  FLESCH  (Arch.f.  mik.  Anat,  1878,  p.  300). 

TAFANI  (Arch.  Ital.  de  Biol,  vi,  p.  207). 

POLITZEE,  "  Die  anatomische  u.  histologische  Zergliederung  d.  mensch- 
lichen  Gehororganes,"  Stuttgart  (Enke),  1889  (see  Zeit.  f.  wiss.  Mik.,  vii, 
3,  1890,  p.  364). 

EICHLEE,  Abh.  d.  math-phys.  Cl.  d.  7c.  Sdchsischen  Ges.  d.  Wiss.,  Bd. 
xviii,  1892,  p.  311  ;  Zeit.  f.  wiss.  MiJc.,  ix,  3,  1893,  p.  380  (detailed 
account  of  manipulations  for  injection  of  blood-vessels  of  the  labyrinth) ; 
SIEBENMANN,  Die  Blutgefdsse  im  Labyrinthe  des  menschlichen  Ohres, 
Wiesbaden,  Bergmann,  1894;  cf.  Zeit.f.  wiss.  Mik.,  xi,  3,  1894,  p.  386. 


Electric  Organs. 

785.  Torpedo.  Raj  a.— BALLOWITZ  (Arch.  mik.  Anat.,  xlii,  1893, 
p.  460)  gives  a  review  of  all  the  older  methods.  He  himself 
gets  the  best  results  by  the  Golgi  impregnation,  controlled 
by  treatment  of  fresh  tissues  for  one  or  two  days  with  osmic 
acid  of  1  per  cent,  and  teasing. 

IWANZOFP  (Bull.  Soc.  Nat.  Moscou,  2,  viii,  1895,  p.  407)  in- 
jects osmic  acid  of  0'5  to  2  per  cent.,  removes  the  pillars  after 
a  few  minutes,  and  hardens  in  2  per  cent,  bichromate  of 
potash,  stains  in  haematoxylin,  and  imbeds  in  paraffin. 

He  (ibid.,  ix,  1895,  p.  74)  fixes  the  organ  in  the  tail  of 
Raja  with  liquid  of  Flemming,  stains,  and  cuts  as  above. 

BALLOWITZ  (Anat.  Hefte,  1  Abth.,  vii,  1897,  p.  285)  finds 
the  method  of  Grolgi  excellent  for  this  organ. 


CHAPTER  XXXIII. 

SOME  OTHER   HISTOLOGICAL  METHODS. 

Connective  Tissues. 

786.  Connective  Tissue. — S.  MAYEE  (Sitzb.  ~k.  Akad.  Wiss., 
Ixxxv,  1882,  p.  69)  recommends  for  staining  fresh  tissue  a 
solution  of  1  gramme  of  "  Violet  B "  (Bindschedler  and 
Busch,  Bale)  in  300  c.c.  of  0*5  per  cent,  salt  solution. 
Elastic  fibres  and  smooth  muscle  also  stain,  but  of  different 
tints. 

For  RANVIER'S  method  of  artificial  oedemata  for  the  study 
of  areolar  tissue,  see  his  Traite,  p.  329. 

FREEBORN  (Amer.  Mon.  Mic.  Journ.,  1888,  p.  231;  Jourti. 
Roy.  Mic.  Soc.j  1889,  p.  305)  recommends  (for  sections)  picro- 
nigrosin,  made  by  mixing  5  c.c.  of  1  per  cent,  aqueous  solu- 
tion of  nigrosin  with  45  c.c.  of  aqueous  solution  of  picric 
acid.  Stain  for  three  to  five  minutes,  wash  with  water,  and 
mount  in  balsam. 

VAN  GIBSON'S  Picro- Saurefuchsin  Stain  has  been  given, 
§289. 

RAMON  Y  CAJAL  recommends  0*1  grm.  of  Saurefuchsin  to 
100  of  saturated  solution  of  picric  acid  (SCHAFFER,  Zeit.  wiss. 
Zool.,  Ixvi,  1899,  p.  214;  Zeit.  f.  wiss.  Mik.,  xvi,  4,  1900, 
p.  464). 

HANSEN  (Anat.  Anz.,  xv,  1898,  p.  152)  adds  5  c.c.  of  2  per 
cent,  solution  of  Saurefuchsin  to  100  c.c.  saturated  solution 
of  picric  acid,  and  for  staining  adds  to  3  c.c.  of  the  mixture 
one  third  of  a  drop  of  2  per  cent,  acetic  acid,  stains  for  a 
few  minutes  or  hours,  rinses  in  3  c.c.  of  water  with  2  drops 
of  the  acidified  stain  added,  dehydrates,  clears  with  xylol, 
and  mounts  in  xylol-balsam.  Connective  tissue  red,  elastin 
and  all  other  elements  yellow. 


436  CHAPTKtt    XXXI 1 1. 

RAMON  Y  CAJAI/S  picro-indigo-carmine  stain  has  been  given, 
§  374. 

EIBBERT  (Zeit.  f.  wiss.  Mik.,  xv,  1898,  p.  93)  recommends  MALLOKT'S 
phospho-molybdic  acid  hsematoxylin  (§  259)  with  previous  mordanting  for 
half  a  minute  in  the  phospho-molybdic  acid,  then  rinsing,  and  staining  for 
five  minutes. 

BENECKE'S  Stain  for  Fibrils  (Verh.  d.  anat.  G-es.,  vii  Vers., 
1893,  p.  165  ;  Zeit.f.  wiss.  Mik.,  xi,  1,  1894,  p.  79)  is  essen- 
tially that  of  KKOMAYER  (§  656). 

See  also  KEOMAYER,  Dermat.  Zeit.,  iii,  1896,  p.  263  ;  Zeit.f.  wiss.  Mik., 
xiv,  1897,  p.  56. 

787.  UNNA  (Monatssch.  f.  prakt.  Dermatol.,  xviii,  1894,  p. 
509  ;  Zeit.  f.  wiss.  Mik.,  xi,  4,  1894,  p.  518)  finds  that  the 
method  of  BKNECKE  (last  §)  is  unsurpassed  for  the  demonstra- 
tion of  collagen  fibrils  alone,  but  prefers  the  following 
whenever  it  is  desirable  to  obtain  at  the  same  time  good 
images  of  the  ground-substance  and  of  other  elements  of 
preparations. 

1.  The  Orcein  Method. — Sections   of   alcohol  material   are 
stained  for  five  minutes  in  Griibler's  strong  solution  of  poly- 
chromatic  methylen  blue.      They  are  then  brought  for  fifteen 
minutes  into  a  neutral  1  per  cent,  solution  of  orcein  in  abso- 
lute alcohol,  rinsed  in  pure  alcohol,  cleared  in  bergamot  oil,, 
jtnd  mounted.      Nuclei  blue,   collagenous    ground- substance 
dark  red,  granules  of  Mastzellen  carmine  red,  protoplasm  of 
Plasmazellen  blue. 

2.  The  Method  of  Sulphosalts. — (a)  Stain  for  five  or  ten 
minutes  in  an  aqueous  2  per  cent,  solution  of  Saurefuchsin, 
rinse,  treat  for  one  or  two  minutes  with  saturated  aqueous 
solution  of  picric  acid,  dehydrate   (two  minutes)  in  absolute 
alcohol  saturated  with  picric  acid,  rinse  with  pure  alcohol, 
clear  and  mount.      (b)   Stain  for  twenty  seconds  in   aqueous 
1    per  cent,    solution   of  Wasserblau,   rinse,   treat    for   five 
minutes  with  neutral  aqueous  1  per  cent,  solution  of  safranin. 
Rinse  and  put  into  absolute  alcohol  until  the  blue  colour  re- 
appears, clear  with  bergamot  oil  and  mount.      Collagen  light 
blue,  nuclei  red. 


SOME    OTHER   HISTOLOGICAL  METHODS.  437 

788.  Adenoid  Tissue.    See  HOEHL,  Arch.  Anat.  Phys.,  Anat.  Abth., 
1897,  p.  133  ;  Zeit.  f.  wiss.  Mik.,  xv,  1898,  p.  228. 

789.  Fat. — DEZHUYSEN   and  FLEMMING  (Zeit.  f.  wiss.  Mile.,  1889, 
pp.  39,  178)  have  discovered  that  fat  that  has  been  stained  black  by  treat- 
ment with  chromo-aceto-osmic  acid  is  dissolved  in  the  course  of  a  few  hours 
in  turpentine,   xylol,  ether,  or  creasote,  and  more  slowly  if  it  has  been 
blackened  with   pure   osmic  acid,   and  FLEMMING   finds  that  very  good 
demonstration  preparations  may  be  made   by  treating  fatty  tissue  with 
chromo-aceto-osmic   acid,    staining    with    safranin   or    gentian,   and  then 
treating  for  a  few  hours  with  turpentine  until  all  the  fat  is  dissolved.     The 
optical   hindrance   caused  by  the    high  refraction  of  the  fat  being  thus 
eliminated,  nuclei  and  cytoplasm  may  be  studied  to  far  greater  advantage 
than  in  the  usual  preparations.     See  also  §  36,  sub  fin. 

790.  Sudan  III  Stain  for  Fat. — DADDI  (Arch.  Ital.  Biol., 
xx vi,  1896,  p.  143)  stains  fat  in  tissues  by  treating  for  5  to 
10  minutes  with  concentrated  alcoholic  solution  of  Sudan 
III,  washing  for  the  same  time  with  alcohol,  mopping  up 
with  blotting-paper,  and  mounting  in  glycerin  (the  speci- 
mens can  hardly  be  mounted  in  balsam  on  account  of  the 
solution  of  the  fat  in  the  absolute  alcohol,  etc.).  Small  fat 
drops  yellow,  large  ones  orange.  The  stain  is  said  to  be 
more  selective  for  fats  than  that  of  osmic  acid. 

Similarly  RIEDER,  see  Zeit.  f.  wiss.  Mik.,  xv,  1898,  p. 
211. 

791.  Granule  cells,  "  Mastzellen,"  "  Plasmazellen,"  and  others. 
For  the  general  characters  of  these  cells,  and  for  EHELICH'S  classification  of 
granules,  see  previous  editions.  I  am  now  much  inclined  to  doubt  the 
objectivity  of  Ehrlich's  classification,  for  the  reasons  assigned  by  FISCHEB 
in  his  Fixirung,  Fdrbung  u.  Bau  des  Protoplasmas,  which  see. 

792.  EHRLICH'S  "Mastzellen"  (Arch.  f.  mik.  Anat.,  xii, 
1876,  p.  263). — The  tissues  must  be  first  well  hardened  in 
strong  alcohol  (chromic  acid  and  its  salts  must  be  avoided). 
They  are  then  placed  for  at  least  twelve  hours  in  a  staining 
fluid  composed  of — 

Absolute  alcohol .  .  .       50  c.c. 

Aqua       ....     100  c.c. 

Acid.  acet.  glacial          .   .  .       12^  c.c. 

— to  which  has  been  added  enough  dahlia  to  give  an  almost 
saturated  solution.  After  staining,  the  preparations  are 
transferred  to  alcohol,  which  washes  out  the  stain  from  all 


438  .  CHAPTER    XXXIII. 

but  the  plasma  cells,  and  may  then  be  mounted  in  resin-tur- 
pentine solution. 

Mucus  cells  and  fat  cells  are  also  sometimes  stained  by 
these  solutions. 

Other  Media.  —  In  a  similar  way  other  soluble  anilins  may 
be  employed  (in  the  form  of  a  fluid  containing  7^  per  cent. 
of  acetic  acid),  —  primula,  iodine  violet,  methyl  violet,  pur- 
purin,  safranin,  fuchsin  ;  of  these,  methyl  violet  gives  the 
best  results. 

See  also  SCHIEFFERDECKER  and  KOSSEL'S  Gewebelehre,  p.  329. 


793.  Plasma  Cells  (NqfrDMANN,  Beitr.  z.  Kenntniss  d.  Mast- 
zellen,  Inauguraldiss.,  Helmstedt,  1884).  —  NOBDMANN  finds  it 
useful  to  employ  a  concentrated  solution  of  vesuvin  contain- 
ing 4  to  5  per  cent,  of  hydrochloric  acid.      Sections  should 
remain  for  a  few  minutes  in  the   solution,  and  then  be  de- 
hydrated with  absolute  alcohol.      The  paper  quoted  contains 
a    detailed    discussion   of    the   micro-chemical    reactions   of 
granule  cells. 

794.  Plasma  Cells  and  Mastzellen.  —  UNNA,  Zeit.f.  wiss.  Mik., 
vii,  4,  1892,  p.  475,  gives  the  following  : 

A.  For  Plasma  Cells. 

Methylen  blue        -r-  .    •  3  c       1*0 

Caustic  potash     itw?.«8  V;  »         0'05 

Distilled  water    •*';:;  .  .     lOO'O 

Add  a  few  drops  of  this  to  ten,  fifty,  or  one  hundred  vols. 
of  anilin  water  (p.  203)  in  a  watch  glass,  and  stain  (alcohol 
material,  or  at  most  sublimate  and  alcohol  material,  not 
chromic  material)  for  half  an  hour,  several  hours,  or  over- 
night. Dehydrate  rapidly  in  absolute  alcohol,  differentiate 
in  creosol  (details  not  given),  rinse  in  xylol,  and  mount  in 
balsam. 

B.  General  Stain,  also  bringing  out  Plasma  Cells. 

,    Methylen  blue       .   .  .  .1*0 

Carbonate  of  potash  .  .1*0 

Distilled  water         '.  .  .     lOO'O 
Alcohol  20-0 


SOME    OTHER    HISTOLOG1CAL    METHODS.  439 

Heat  on  a  water- bath  until  reduced  to  lOO'O.  Use  for 
staining  undiluted,  or  diluted  with  one  vol.  of  anilin  water. 
Differentiate  (details  not  given)  with  glycol,  styron,  or  creosol. 
Mastzellen  are  not  differentiated. 


c.  Stain  giving  Red  Mastzellen  with  Blue  Plasma  Cells. 

Methylen  blue          .  ,  .'.         1-0 

Kali  Carbon,  (natron  carbon,  ammon. 

carbon)  .  .  '.  .         1*0 

Aq.    dest.    (Aq.    carbolisata,    chloro- 

forma)     .  .,  .  .     10OO 

Dilute  about  1 00-fold,  stain  slowly,  treat  with  70  to  80  per 
cent,  alcohol,  differentiate  in  styron,  and  bring  through  ber- 
gamot  oil  or  xylol  into  balsam.  In  this  process  the  granules 
of  the  Mastzellen  stain  red  in  consequence  of  the  formation 
of  methyl  en  red  in  the  staining  bath. 

See  further  UNNA  in  Monatssch.  f.  prdkt.  Dermatol.,  xii,  1891,  p.  296 ; 
Zeit.f.  wiss.  Mik.,  ix,  1,  1892,  p.  92,  and  Monatssch.  f.  prakt.  Dermatol., 
xix,  1894,  p.  225 ;  Zeit.  f.  wiss.  Mik.,  xii,  2,  1895,  p.  58  ;  also  VAN  DEE 
SPEK  and  UNNA,  loc.  cit.,  xiii.  1891,  p.  364 ;  Zeit.  f.  wiss.  Mik.,  xi,  1,  1892, 
p.  89. 

795.  Plasma  Cells  and  Mastzellen. — BKKGONZINI  (Anat.  Anz., 
vi,  1891,  pp.  595—600;  Zeit.f.  wiss.  Mik.,  ix,  1,  1892,  p.  95). 
— Mix  1  volume  of  0'2  per  cent,  solution  of  Saurefuchsin 
with  2  vols.  of  a  like  solution  of  methyl  green,  and  2  vols. 
of  a  like  solution  of  gold-orange,  and  filter  through  cotton 
wool.  Stain  alcohol  or  sublimate  sections  (after  washing  in 
water)  for  three  to  four  minutes,  wash  for  one  or  two 
minutes  in  water,  bring  into  absolute  alcohol  for  two  minutes, 
clear  in  bergamot  oil  or  pure  creosote,  wash  in  turpentine, 
and  mount  in  balsam. 

One  sort  of  gold-orange  precipitates  methyl  green,  and 
therefore  cannot  be  used  in  this  mixture.  Orange  G  may 
be  used  instead. 

796.  Plasma  Cells.— JADASSOHN  (Arch.  f.  Dermatol.  u.  Syphilis, 
Erganzungsheft  1,  1892,  p.  58 ;  Zeit.  f.  wiss.  Mik.,  ix,  2,  1892,  p.  226)  re- 
commends staining  for  not  too  long  in  a  1  :  2000  strongly  alkaline  or  borax 
solution  of  thionin,  and  washing  out  with  acidulated  water. 

See  also  VON  MABSCHALKO,  Arch.  f.  Dermatol.  u.  SyphiL,  xxx,  1895, 


440  CHAPTER  XXXIII. 

1>.  3;  Zeit.f.  wiss.  Mik.,  xii,  1,  1895,  p.  64,  and  KEOMPECHEE,  ibid.,  xv, 
1899,  p.  458. 

797.  Clasmatocytes,  KANVIEK  (C.  R.  Acad.  des  Sci.,  1890). 
— A   piece    of   suitable  membrane    (epiploon   of   mammalia, 
mesentery  of  batrachia)   is    stretched   secundum  artem  on  a 
slide,  and  a  few  drops  of  1  per  cent,  solution  of  osmic  acid 
are  allowed  to  fall  on  to  it.      After  one  or  two  minutes  it  is 
washed  with  water  and  stained  with  concentrated  aqueous 
solution  of  methyl  violet  5  B  diluted  with  ten  parts  of  dis- 
tilled water.      Glycerin  may  be  added  to  make  the  prepara- 
tion permanent,  but  does  not  succeed  very  well,  as  it  causes 
the  stain  to  diffuse.      Brunts  glucose  medium  (§  421)  would 
probably  be  preferable. 

798.  Elastic  Tissue. — Two  of  the  most  salient  characters  of 
elastic  fibres  are  that  they  have  a  great  affinity  for  osmium, 
staining  with  much  more   rapidity  than  most  oth'er  tissue - 
elements,  and  that  they  are  not  changed  by  caustic  soda  or 
potash.       A  further   character   is   that   they   have   a   great 
affinity  for  certain  anilin  dyes,  especially  Victoria  blue. 

•'  For  a  review  of  the  older  methods  of  BALZEE,  UNNA, 
LUSTGAETKN,  and  HERXHEIMER,  see  the  paper  by  G.  MARTINOTTI 
in  Zeit.f.  wiss.  Mik.,  iv,  1,  1887,  p.  31. 

The  method  of  LUSTGAETEN  has  been  given  in  §  275.  The 
Colour  used  by  him  was  called  "  Yictoriablau  4  A  /'  and  this 
may  be  important. 

The  method  of  G.  MAETINOTTI  (loc.  cit.)  is  as  follows  : — Fix 
in  a  chromic  liquid,  wash,  stain  for  forty-eight  hours  in 
strong  (5  per  cent.  Pfitzner's)  solution  of  safranin,  wash, 
dehydrate,  clear,  and  mount  in  balsam.  Elastic  fibres  are 
stained  an  intense  black,  the  rest  of  the  preparation  showing 
the  usual  characters  of  a  safranin  stain. 

The  staining  will  be  performed  quicker  if  it  be  done  at  the  temperature 
of  an  incubating  stove  (GEIESBACH,  ibid.,  iv,  1887,  p.  442).  And  FERKIA 
(ibid.,  v,  3,  1888,  p.  342)  says  that  clearer  preparations  will  be  obtained  if 
the  sections  be  left  for  a  long  time,  say  twenty-four  hours,  in  the  alcohol, 
or  be  treated  for  a  short  time  with  very  dilute  alcoholic  solution  of  caustic 
'  potash.  This  decolourises  more  completely  the  ground  of  the  preparations. 

Another  safranin  method,  which  seems  to  have  the  fault  of  requiring  a 
too  minute  attention  to  details,  is  that  of  MIBELLI,  see  Mon.  zool.  italiano, 
1,  p.  17,  or  Zeit.f.  wiss.  Mik.,  vii,  2,  1890,  p.  225  (the  report  in  Journ.  Roy. 
Mic.  Soc.,  1890,  p.  803,  is  vitiated  by  a  misprint). 


SOME    OTHKR    HISTOLOGICAL    METHODS.  441 

For  the  elastic  tissue  of  the  skin,  see  PASSABGE  and  KBOSING,  Dermat. 
Stud.,  xviii,  1894. 

See  also  for  staining  and  dissociation  AGABABOW,  Arch.  mik.  Anat.,  1, 
1897,  p.  566,  et  seq. 

For  C.  MABTINOTTI'S  silver  impregnation,  see  Zeit.f.  wiss.  Mik.,  v,  1888, 
p.  521,  or  Arch.  Hal.  BioL,  xi,  1889,  p.  257. 

SCHUMACHER  (Arch.  mik.  Anat.,  lv,  1899,  p.  151,  Zeit.f.  wiss.  Mik.,  xvi, 
4,  1900,  p.  456)  has  had  good  results  (for  the  spleen)  with  a  mixture  of  1 
part  1  per  cent,  nigrosin  and  9  parts  saturated  aqueous  picric  acid. 

See  also  §  821. 

799.  UNNA'S    Modified  Orcein  Method  (Monatssch.  f.  prakt. 
DermatoL,  xix,  1894,  p.  397 ;  Zeit.  f.  wi*8.  Mile.,  xii,  2,  1895, 
p.  240). — The  following  solution  is  made  :  Griibler's  orcein 
1  part,  hydrochloric  acid  1  part,  absolute  alcohol  100  parts. 
The    sections   are   put    into    a   porcelain   capsule   with    just 
enough  of  the  stain  to  cover  them,  and  the  whole  is  warmed 
in  a  stove  or  over  a  naked  Hame  to  about  30°  C.      After  ten 
to  fifteen  minutes  the  stain  becomes    quite  thick,  owing  to 
evaporation   of   the   alcohol.       The    sections   are   then   well 
rinsed  in  alcohol,  cleared,  and  mounted.      Elastin  dark  brown, 
collagen  light  brown. 

For  UNNA'S  earlier  orcein  method,  see  third  edition,  or  Monatssch.  f. 
prakt.  DermatoL,  xii,  1891,  p.  394  (Zeit.f.  wise.  Mik.,  ix,  1,  1892,  p.  94). 

See  also  ZENTHOEFEB,  in  Unna's  DermatoL  Studien,  1892,  or  Zeit.  f. 
wiss.  Mik.,  ix,  4,  1893,  p.  509 ;  KOPPEN,  Zeit.  f.  wiss.  Mik.,  vi,  4,  1889, 
p.  473 ;  and  vi,  1,  1890,  p.  22,  or  third  edition  ;  BUBCI,  Journ.  Eoy.  Mic. 
Soc.,  1891,  p.  831,  and  1892,  p.  292,  or  third  edition ;  HANSEN,  Virchow's 
Archiv,  cxxxvii,  1894,  p.  25;  Zeit.  f.  wiss.  Mik.,  xi,  3,  1894,  p.  383  i 
KULTSCHIZKT,  ibid.,  xiii,  1,  1896,  p.  74,  or  the  original,  Arch.f.  mik.  Anat., 
xlvi,  1895,  p.  673  ;  GUNTHEB,  Zeit.  f.  wiss.  Mik.,  xiii,  1896,  p.  230 ; 
SCHIEFFEBDECKEB,  ibid.,  p.  302  ;  TfiiEPEL,  ibid.,  xiv,  1897,  p.  31 ;  LOISEL, 
Journ.  de  I'AnaL,  et  de  la  Phys.,  xxxiii,  1897,  p.  134;  GABDNEB,  BioL 
Centralb.,  xvii,  1897,  p.  398 ;  LIVINI,  Monitore  Zool.  ItaL,  vii,  1896,  p.  45 
(Journ.  Roy .  Mic.  Soc.,  1899,  p.  455). 

800.  WEIGERT'S  Fuchsin-resorcin  Method  (Zeit.  f.  wiss.  Hik., 
xvi,   1899,  p.  81). — 1  per  cent,  of  basic  fuchsin  and  2  per 
•cent,    of    resorcin    (or    of    carbolic   acid)    are    dissolved    in 
water.      200  c.c.  of  the  solution  are  raised  to  boiling  point  in 
a  capsule,  and  25  c.c.  of  Liquor  Jerri  set*  quick  lor  ati  P.  Gr.  are 
added,  and  the  whole  is  boiled,  with  stirring,  for  two  to  five 
minutes  more.      A  precipitate  is  formed.      After  cooling  the 
liquid   is  filtered,  and  the  precipitate  which  remains  on  the 


442  CHAPTER  XXXIII. 

filter  is  brought  back  into  the  capsule,  and  there  boiled  with 
200  c.c.  of  94  per  cent,  alcohol.  Allow  to  cool,  filter,  make 
ujp  the  filtrate  to  200  c.c.  with  alcohol,  and  add  4  c.c.  of 
hydrochloric  acid. 

Stain  sections  (of  material  fixed  in  any  way)  for  twenty 
minutes  to  an  hour,  wash  with  alcohol,  clear  with  xylol  (not 
with  an  essence).  Elastic  fibres  dark  blue  on  a  light  ground. 
Nuclei  generally  unstained  ;  they  may  be  after-stained  with 
carmine,  etc. 

Bone* 

801.  Bone,  Non-decalcified  (KANVIEE,  Traite,  p.  297).— 
RANVIER  points  out  certain  precautions  that  it  is  necessary  to 
take  in  the  preparations  of  sections  of  dry  bone.  In  general 
the  bones  furnished  by  "  naturalists  "  or  procured  in  ana- 
tomical theatres  contain  spots  of  fatty  substance  that  prevent 
good  preparations  from  being  made.  Such  spots  are  formed 
when  bones  are  allowed  to  dry  before  being  put  into  water 
for  maceration  ;  when  a  bone  is  left  to  dry  the  fat  of  the 
medullary  canals  infiltrates  its  substance  as  fast  as  its  water 
evaporates. 

Bones  should  be  plunged  into  water  as  soon  as  the  sur- 
rounding soft  parts  have  been  removed,  and  should  be 
divided  into  lengths  with  a  saw  whilst  wet.  The  medulla 
should  then  be  driven  out  from  the  central  canal  by  means 
of  a  jet  of  water;  spongy  bones  should  be  submitted  to 
hydrotomy  as  follows  :  — An  epiphysis  having  been  removed, 
together  with  a  small  portion  of  the  diaphysis,  a  piece  of 
caoutchouc  tubing  is  fixed  by  ligature  on  to  the  cut  end 
of  the  diaphysis,  and  the  free  end  of  the  piece  of  tubing 
adapted  to  a  tap  through  which  water  flows  under  pressure. 

As  soon  as  the  bones,  whether  compact  or  spongy,  have 
been  freed  from  their  medullary  substance  they  are  put  to 
macerate.  The  maceration  should  be  continued  for  several 
months,  the  liquid  being  changed  from  time  to  time.  As 
soon  as  all  the  soft  parts  are  per-fectly  destroyed,  the  bones 

*  For  a  minutely  detailed  review  (40  pages,  with  references  to  80 
memoirs)  of  the  whole  subject  of  the  technique  of  bone,  see  the  paper  of 
SCHAFFEB,  Die  Methodik  der  histologischen  Untersuchung  des  Knochen- 
gewebes,  in  Zeit.  f.  wiss  Mik.,  x,  2,  1893,  p.  167. 


SOME   OTHER   HISTOLOGICAL    METHODS.  443 

may  be  left  to  dry.  When  dry,  they  should  be  of  an  ivory 
whiteness,  and  their  surfaces  exposed  by  cutting  of  a  uniform 
dulness. 

Thin  sections  may  then  be  cut  with  a  saw  and  prepared  by 
rubbing  down  with  pumice-stone.  Compact  pumice-stone 
should  be  taken  and  cut  in  the  direction  of  its  fibres.  The 
surface  should  be  moistened  with  water  and  the  section  of 
bone  rubbed  down  on  it  with  the  fingers.-  When  both  sides 
of  the  sections  have  been  rubbed  smooth  in  this  way,  another 
pumice-stone  may  be  taken,  the  section  placed  between  the 
two,  and  the  rubbing  continued.  As  soon  as  the  section  is 
thin  enough  to  be  almost  transparent  it  is  polished  by 
rubbing  with  water  (with  the  fingers)  on  a  Turkey  hone  or 
lithographic  stone.  Spongy  bone  should  be  soaked  in  gum 
and  dried  before  rubbing  down  (but  see  VON  KOCH'S  copal 
process,  ante,  §  172,  and  EHRENBAUM'S  colophonium  process, 

§ 


For  the  process  of  WEIL  for  bone  and  teeth,  see  §  175. 

ROSE  (Anat.  Anz.,  vii,  1892,  pp.  512—519  ;  Zeit.  f.  wiss.  Mile.,  ix,  4, 
1893,  p.  506)  follows  Koch's  process.  He  penetrates  first  with  a  mixture 
of  cedar  oil  and  xylol,  then  with  pure  xylol,  and  imbeds  in  solution  of 
damar  in  chloroform  or  xylol.  The  method  can  be  combined  with  Golgi's 
impregnation. 

WHITE  (Jo  urn.  Roy.  Mic.  Soc.,  1891,  p.  307)  recommends 
the  following  :  —  Sections  of  osseous  or  dental  tissue  should 
be  cut  or  ground  down  moderately  thin,  and  soaked  in  ether 
for  twenty-four  hours  or  more.  They  should  then  be  put 
for  two  or  three  days  into  a  thin  solution  of  collodion 
stained  with  fuchsin  (made  by  dissolving  the  dye  in  methy- 
lated spirit,  adding  the  requisite  quantity  of  ether,  then  the 
pyroxylin)  .  The  sections  are  then  put  into  spirit  to  harden 
the  collodion.  After  this  they  are  ground  down  to  the 
requisite  thinness  between  two  plates  of  old  ground  glass, 
with  water  and  pumice  powder,  and  mounted,  surface  dry,  in 
stiff  balsam  or  styrax,  care  being  taken  to  use  as  little  heat 
as  possible.  Lacunae,  canaliculi,  and  dentinal  tubuli  are  found 
infiltrated  by  the  coloured  collodion. 

MATSCHINSKY  (Arch.  f.  mik.  Anat.,  xxxix,  1892,  p.  151,  and  xlvi,  1895, 
p.  290  ;  Zeit.  f.  wiss.  Mik.,  ix,  3,  1893,  p.  353,  and  xiii,  1896,  p.  68)  after 
grinding,  impregnates  with  nitrate  of  silver. 


444  CHAPTER   XXXIII. 

For  a  similar  method  of  KUPRECHT,  see  Zeit.  /.  wiss.  Mile.,  xiii,  1, 1896, 
•p.  21,  wherein  see  also  quoted  (p.  23)  a  method  of  ZIMMEEMANN. 


802.  Sections  of  Bones  or  Teeth  showing  the   Soft  Parts.— 
NEALEY  (Amer.  Mo  it.  Mic.  Journ.,  1884,  p.  142  ;   Journ.  Roy. 
Mic.  Soc.,  1885,  p.  348)  says  that  perfectly  fresh  portions  of 
bone  or  teeth  may  be  ground  with  emery  on  a  dentist's  lathe, 
;and  good  sections,  with  the   soft  parts  in  situ,  obtained  in 
half  an  hour. 

HOPKWELL-SMITH  (Journ.  Brit.  Dent.  ASK.,  xi,  1890,  p.  310  ; 
Journ.  Roy.  Mic.  Soc.,  1890,  p.  529)  says  that  for  preparing 
sections  of  teeth  showing  odontoblasts  in  situ  the  best  plan  is 
to  take  embryonic  tissues.  A  lower  jaw  of  an  embryonic 
kitten  or  pup  may  be  taken,  and  hardened  in  solution  of 
Miiller  followed  by  alcohol,  then  cut  with  a  freezing  micro- 
tome. The  knife  cuts  quite  easily  the  thin  cap  of  semi- 
calcified  dentine  and  bone. 

WEIL  (loc.  cit.,  §  175)  fixes  pieces  of  fresh  teeth  in  subli- 
mate, stains  with  borax-carmine,  brings  them  through 
alcohol  into  chloroform  and  chloroform- balsam,  and  after 
hardening  this  by  heat  proceeds  to  grind  as  usual  (§  172). 

See  also  EOSE,  last  §. 

803.  VIVANTE  (Intern.  Monatssch.  f.  Anat.  u.  Phys.,  ix,  1892,  p.  394; 
Zeit.  f.  wiss.  Mile.,  ix,  3,  1893,  p.  351)  has  made  out  that  thin  specimens  of 
bone  can  be  successfully  treated  by  Golgi's  bichromate  of  silver  process.    He 
places  portions  of  frontal  bone  of  four  to  six  months  calves,  which  are  not 
more  than  3  to  4  mm.  thick,  for  eight  days  in  solution  of  Miiller,  th.en  in  the 
•osmium  bichromate  mixture,  and  then  in  the  silver  solution.     After  impreg- 
nation the  specimens  should  be  decalcified,  which  may  be  done  by  putting 
them  for  twenty  days  into  von  Ebner's  mixture  (§  563),  after  which  they 
•should  be  well  washed  with  water  and  brought  into  solution  of  carbonate  of 
soda,  and  finally  imbedded  in  paraffin.     Tor  his  quinolein  blue  method,  see 
last  edition. 

For  UNDERWOOD'S  gold  process  for  teeth,  and  for  that  of  LEPKOWSKY, 
•see  third  edition. 

For  the  study  of  the  vessels  in  teeth,  LKPKOWSKY  (Anat. 
Hefte,  viii,  1897,  p.  568)  injects  with  Berlin  blue,  hardens 
the  teeth  with  a  piece  of  the  jaw  for  one  or  two  days  in  50 
per  cent,  formol,  decalcifies  in  10  per  cent,  nitric  acid  (eight 
to  fourteen  days,  change  frequently)  and  makes  celloidin 
sections. 


SOME    OTHER    HISTOLOGICAL    METHODS.  445 

804.  Bone,  Decalcified  (FI.EMMING,  Zeit.  f.  wiss.  Mik.,  1886,, 
p.  47). — Sections  of  decalcified  bone  are  soaked  in  water,  and 
brought  in  a  drop  of  water  on  to  a  glass  plate,  where  they 
are   spread  out  flat.      The  excess  of  water  is  removed  with 
blotting-paper,  and  the  sections  are    covered  with   another 
glass  plate,  to  prevent  them  from  rolling.       The   whole    is 
brought  into  a  plate  and  covered  with  alcohol.      After  the 
lapse  of  half  an  hour  the  sections  have  become  fixed  in  the 
flat   position,    and   may   be    brought    into   absolute    alcohol 
without  risk  of  their  rolling.      To  mount  them,  wash  them 
with  fresh  alcohol  (which  may  be  followed  by  ether)  ;   lay 
them  again  flat  on  glass,  and  cover  them  with  a  double  layer 
of   blotting-paper   and   a   somewhat   heavy  glass  plate,  and 
let  them  dry  for  a  day  in  the  air  or  in  a  stove.      When  they 
are  dry,  put  a  drop  of  melted  balsam  on  a  slide,  and  let  it 
spread  out  flat  and  cool.      Prepare  a  thin  glass  cover  in  the 
same   way,  put  the  section  on  the  prepared  slide,  cover  it 
with  the  prepared  cover,  put  on  a  clip,  and  warm. 

By  this  process  sections  can  be  very  expeditiously  pre- 
pared, which  show  the  lacunar  system  injected  with  air  in 
quite  as  instructive  a  manner  as  non-decalcified  sections. 

805.  Stains  for  Cartilage   (and  Decalcified  Bone).— For  an 
excellent  discussion    (especially  as  regards    staining)   of  the 
methods    for    these    objects,  see    the    exhaustive    paper    of 
SCHAFFER  in  Zeit.  f.  wiss.  Mik.,  v,   1,   1888. 

KOLLIKER  (Zeit.  f.  wiss.  Zool.,  xliv,  1886,  p.  662)  recom- 
mends the  following  for  the  demonstration  of  the  fibres  of 
Sharpey  in  decalcified  bone.  Sections  are  treated  with  con- 
centrated acetic  acid  until  they  become  transparent,  and  are 
then  put  for  one  quarter  to  one  minute  into  a  concentrated 
solution  of  indigo-carmine,  then  washed  in  water  and  mounted 
in  glycerin  or  balsam.  In  successful  preparations  the  fibres 
of  Sharpey  appear  stained  of  a  pale  or  dark  red,  the  remain- 
ing bone  substance  blue. 

ZACHABIADES  (Zeit.f.  wiss.  Mile.,  x,  4, 1893,  p.  447)  has  the  following : — 
Bone  is  decalcified  bj  means  of  picric  acid,  washed,  and  put  into  alcohol  and 
sectioned.  The  sections  are  placed  on  a  slide  and  treated  for  a  few  seconds 
with  1  per  cent,  solution  of  osmic  acid.  They  are  then  stained,  either  for 
twenty-four  hours  in  a  weak  aqueous  solution  of  quinolein  blue,  or  for  a  few 
minutes  in  a  saturated  aqueous  solution  of  safranin.  They  are  then  treated 


446  CHAPTEE    XXX J II. 

on  a  slide  with  a  drop  of  40  per  cent,  solution  of  caustic  potash,  the  slide 
being  warmed  over  a  flame  until  the  sections  spread  out  flat.  The  excess  of 
potash  is  then  removed  and  the  sections  are  carefully  washed  with  water, 
covered,  and  examined.  The  safranin  preparations  may  be  permanently  pre- 
served in  glycerin  containing  a  small  proportion  of  safranin. 

SCHAFFER'S  safranin  method  (Zeit.  f.  wix*.  Mile.,  v,  1888,, 
p.  17,  modified  from  BOUMA,  Centralb.f.  d.  med.  Wis*.,  1883, 
p.  866). — Sections  of  bone  decalcified  with  nitric  acid  (chromic 
acid  may  be  used,  but  the  stain  will  be  less  brilliantly  con- 
trasted) are  stained  for  half  an  hour  to  one  hour  in  0'05  per 
cent,  aqueous  solution  of  safranin,  washed  with  water,  put 
for  two  or  three  hours  in  O'l  per  cent,  solution  of  corrosive 
sublimate,  and  examined  in  glycerin.  In  order  to  make 
permanent  preparations,  the  sections  on  removal  from  the 
sublimate  are  rinsed  with  alcohol,  pressed  on  to  a  slide  with 
filter-paper,  cleared  for  a  long  time  in  bergamot  oil  or  clove 
oil,  and  mounted  in  xylol  balsam. 

This  is  a  double  stain  ;  cartilage,  orange  ;  bone,  uncoloured 
(or  green  in  chromic  objects)  ;  marrow,  red. 

BAYERI/S  method  for  ossifying  cartilage  (Arch.f.  mik.  Anat., 
1885,  p.  35)  is  as  follows  : — Portions  of  ossified  cartilage 
are  decalcified  as  directed  §  578,  cut  in  paraffin,  stained  in 
MerkePs  carmine  and  indigo -carmine  mixture,  §  '372,  and 
mounted  in  balsam. 

Aqueous  solution  of  benzoazurin  has  been  commended  as 
•a  stain  for  ossifying  cartilage  by  ZSCHOKKE,  see  Zeit.  f.  wis*. 
Mik.,  x,  3,  1893,  p.  381. 

A  process  recommended  by  BAUMGARTEN  has  been  given, 
§  375. 

MOERNER  (Skandinavisches  Arch.f.  Physiol.,  i,  1889,  p.  216  ;  Zeit.f. 
wiss.  Mik.,  vi,  4,  1889,  p.  508)  gives  several  stains  for  tracheal  cartilage, 
chiefly  as  microchemical  tests,  for  which  see  third  edition. 

See  also  a  critique  of  these  methods  by  WOLTERS  in  Arch.f.  mik.  Anat., 
xxxvii,  1891,  p.  492;  Zeit.f.  wiss.  Mik.,  viii,  3,  1891,  p.  383  ;  and  on  the 
whole  subject  of  cartilage  see  SCHIEFFERDECKER'S  Gewebelehre,  p.  331. 

FDSARI  (Arch.  Ital.  BioL,  xxv,  1896,  p.  200 ;  Zeit.  f.  wiss. 
Mik.,  xiii,  1896,  p.  488)  makes  sections  of  fresh  cartilage, 
puts  them  for  twenty-four  hours  into  1  per  cent,  nitrate  of 
silver,  washes,  dehydrates,  and  exposes  to  the  light  in 
iDalsam. 


SOME    OTHER   HISTOLOGICAL  METHODS.  447 


Blood. 

806.  Introduction. — The  technique  of  blood  is  most  elabo- 
rate ;   see,  for  instance,  the  voluminous  work  of  HAY  EM,  Da 
sang  et  de  ses  alterations  anafomiques,   pp.   1035,  with    126 
figures,  Paris,  Masson,  1889   (a  report  of  ove'r  twenty  pages 
on  this  important  work  is  contained  in  Zeit.  f.  wisx.  Mik., 
vi,    3,    1889,   p.   330,    et  seq.)  •   COLES,    The    Blood  :   how   to 
examine  and  diagnose  its  diseases,  London,  Churchill,  1898, 
260  pp.,  and  6  pis.  ;   as  well   as  the  numerous  memoirs  of 
LOWIT,  EHKLICH,  and  others   (see  the  bibliography  in  GIGLIO- 
Tos,  Mem.  Accad.  Torino,  xlvii,  1897,  p.  37  ;   also  previou* 
editions) . 

I  confine  myself  to  giving  a  few  methods  that  may  be 
useful  to  the  general  student. 

807.  Fixing  and  Preserving  Methods. — The  school  of  Ehrlich 
fix  by  heat.      A  film  of  blood  is  spread  on  a  cover-glass  and 
allowed    to   dry   in   the   air,  and  then  fixed   by  passing  the 
cover  a  few  times,  three  to  ten  or  twenty,  through  a  flame, 
or   by  laying  it  face  downwards   on   a   hot  plate   kept   for 
several  minutes  or  as  much  as  two  hours  at  a  temperature 
at  which  water  not  only  boils  but  assumes   the  spheroidal 
state    (110°  to  150°  C.).      For  details  see  GULLAND,  Scottish 
Med.  Journ.,  April,  1899,  p.  312  ;   KUBINSTKIN,  Zp.it.  f.  wiss. 
Mik.,  xiv,  1898,  p.  456 ;  ZIELINA,  ibid.,  p.  463  ;  Journ.  Roy. 
Mic.    Soc.,    1898,    pp.    488,    489.      The   method    is,   in    my 
opinion,  unutterably  barbarous,  and  should  be  abandoned  in 
favour  of  wet  methods. 

In  wet  methods,  either  the  blood  is  mixed  at  once,  on 
being  drawn,  with  some  fixing  and  preserving  medium,  and 
studied  as  a  fluid  mount ;  or  cover-glass  films  are  prepared 
and  put  into  a  fixing  liquid  before  the  film  has  had  time  to 
dry. 

MUIR  (Journ.  of  Anat.  and.  Phys.,  xxvi,  1892,  quoted  from 
Gulland,  below)  makes  cover- glass  films  and  drops  them 
into  saturated  sublimate  solution,  and  after  half  an  hour 
washes,  dehydrates,  and  passes  through  xylol  into  balsam. 

GULLAND  (Brit.  Med.  Journ.,  March  13th,  1897;  Scottish 
Med.  Journ.,  April,  1899)  makes  cover-glass  films,  and  after 


448  CHAPTER    XXX HI. 

a  few  seconds   drops  them  face   downwards  into  a  solution 
of— 

Absolute  alcohol  saturated  with  eosin      .      25  c.c. 
Pure  ether  .  .  .  .  .      25    „ 

Sublimate   in   absolute   alcohol    (2  grms. 

to  10  c.c.)    .  .  .  .  .5  drops. 

After  three  or  four  minutes  they  are  washed,  stained  for 
one  minute  in  saturated  aqueous  solution  of  methylen  blue, 
or  in  hsemalum,  washed,  dehydrated,  and  passed  through 
xylol  into  balsam.  Staining  with  Ehrlich's  triacid  also 
gives  good  results. 

JENNEE  (Lancet,  1899,  No.  6,  p.  370;  Journ.  Roy.  Mic. 
Soc.,  1899,  p.  231)  mixes  equal  parts  of  1'2  to  1'25  per  cent, 
water-soluble  eosin  (Griibler)  and  I  per  cent,  methylen  blue,, 
niters  after  twenty-four  hours,  washes  the  precipitate  on  the 
filter,  dries  it,  dissolves  it  in  200  parts  of  pure  methyl 
alcohol,  and  puts  cover-glass  preparations  into  the  solution, 
in  which  they  are  fixed  and  stained  in  three  minutes.  (The 
above  mentioned  precipitate  is  a  "  neutral "  colour,  see 
p.  194.) 

Or,  simply  mix  125  c.c.  of  0'5  per  cent,  solution  of  the 
eosin  in  methyl  alcohol  with  100  c.c.  of  0*5  per  cent,  solution 
of  methylen  blue. 

WEEMEL  (Zeit.  f.  wiss.  Mile.,  xvi,  1899,  p.  50 ;  Journ.  Roy.  Mic.  Soc., 

1899,  p.  452)  gives  some  extremely  complicated  methods  of  fixing  and  stain- 
ing in  solutions  of  methylen  blue,  eosin,  etc.,  in  formol,  which  I  am  not  sure 
that  I  rightly  understand. 

See,  for  the  action  of  formaldehyde  on  red  corpuscles,  and  for  some  formol 
mixtures,  MABCANO,  Arch.  d.  Med.  Exper.,  xi,  1899,  p.  434 ;  Zeit.f.  wiss. 
Milt.,  xvi,  3,  1899,  p.  364;  also  a  method  of  KIZEE,  Journ.  Roy.  Mic.  Soc., 

1900,  p.  128. 

Most  recent  authors  are  agreed  that  by  far  the  most  faith- 
ful fixing  agent  for  blood -corpuscles  is  osmic  acid.  A  drop 
or  two  of  blood  (BiONDi  recommends  two  drops  exactly)  is 
mixed  with  5  c.c.  of  osmic  acid  solution,  and  allowed  to 
remain  in  it  for  from  one  to  twenty-four  hours.  The  exact 
degree  of  concentration  of  the  osmic  acid  solution  is  a 
somewhat  important  point,  and  must  be  made  out  by  experi- 
ment for  each  form.  As  a  rule  it  should  be  strong,  1  to  2 
per  cent.  According  to  BIONDI,  2  per  cent,  is  best.  Fixed 
specimens  may  be  preserved  for  use  in  acetate  of  potash 


SOME    OTHER   HISTOLOGICAL    METHODS.  449 

solution  (MAX  FLESCH,  Zeit.  f.  wiss.  Mik.,  v,  1,  1888, 
p.  83). 

GRIESBACH  also  (op.  cit.,  p.  328)  prefers  osmic  acid,  not 
only  as  being  a  first-rate  fixing  agent,  but  because  it  can  be 
combined  with  certain  stains  without  decomposing  them. 
He  mentions  methyl  green,  methyl  violet,  crystal  violet, 
safranin,  eosin,  Saurefuchsin,  rhodamin,  and  iodine  in  potassic 
iodide. 

Eossi  (Zeit.  f.  wiss.  Mik.,  vi,  4,  1889,  p.  475)  advises  a 
mixture  of  equal  parts  of  1  per  cent,  osmic  acid,  water,  and 
strong  solution  of  methyl  green,  permanent  mounts  being 
made  by  means  of  glycerin  cautiously  added. 

EWALD  (Zeit.  Biol.,  xxxiv,  1897,  p.  257)  mixes  three  to 
four  drops  of  blood  of  amphibia  or  reptiles  with  10  c.c.  of  a 
solution  of  0*5  per  cent,  osmic  acid  in  0'5  per  cent,  salt 
solution  (for  mammals  0*6  to  0*7  per  cent,  salt),  siphons  off  the 
supernatant  liquid  after  twenty-four  hours  with  his  capillary 
siphon  (§  3,  p.  4),  and  substitutes  water,  alum-carmine,  etc., 
and  lastly,  50  per  cent,  alcohol. 

See  also  ABNOLD,  Arch.  path.  Anat.,  cxlviii,  1897,  p.  479. 

The  mercurial  liquids  of  Pacini  (§  405)  used  to  be  con- 
sidered good.  HAYEM  (op.  cit.;  see  also  Zeit.  f.  wiss.  Mik., 
vi,  3,  1889,  p.  335)  has  the  following  formula  :  sublimate 
0'5,  salt  1,  sulphate  of  soda  5,  and  water  200.  This  should 
be  mixed  with  blood  in  the  proportion  of  about  1  :  100. 
Eosin  may  be  added  to  it.  Lowrr's  formula  (Sitzb.  k.  Akad. 
Wiss.  Wien,  xcv,  3,  p.  129  ;  Zeit.  wiss.  Mik.,  vi,  1,  1889, 
p.  75)  consists  of  5  c.c.  cold  saturated  sublimate  solution,  5 
grms.  sulphate  of  soda,  2  grms.  salt,  and  300  c.c.  water. 
Mosso  finds,  however,  that  both  of  these  are  too  weak  in 
sublimate. 

Of  course  other  well-tried  fixing  fluids,  such  as  Flemming's 
solution,  or  Hermann's,  may  also  be  used  for  blood. 

LAYDOWSKY  (Zeit.  f.  wiss.  Mik.,  x,  1,  1893,  p.  4)  describes  some  remark- 
able results  obtained  by  fixing  with  2  per  cent,  iodic  acid,  and  staining  with 
Neu-Victoriagriin,  methyl  violet  6s,  or  gentian  violet,  a  process  which  is 
said  to  reveal  the  presence  of  nuclei  in  elements  generally  considered  to  be 
apyrenematous. 

DUBOSCQ  (Arch.  Zool.  Exper.,  vi,  1899,  p.  481  ;  Journ. 
Roy.  Mic.  Soc.,  1899,  p.  545)  uses  (for  blood  of  Chilopoda) 

29 


450  CHAPTER   XXXIII. 

a  solution  of  acetic  acid,  copper  ac'etate,  copper  chloride, 
osmic  acid,  thionin,  1  grm.  each,  water  400,  which,  mixed 
with  the  blood,  fixes  and  stains  in  about  two  minutes. 

808.  Stains  for  Blood. — Blood  prepared  as  above  can  be 
satisfactorily  stained  with  many  of  the  usual  reagents. 

Eosin  stains  rose -red  all  parts  of  blood- corpuscles  that  con- 
tain haemoglobin  (see  WISSOZKY,  Arch.  f.  mik.  Anat.,  1876, 
p.  479). 

WISSOZKY  (loc.  cit.)  stains  in  a  solution  of  equal  parts  of 
eosin  and  alum  in  200  parts  of  alcohol,  and  then  with  hsema- 
toxylin. 

MOORE  (The  Microscope,  1882,  p.  73;  Journ.  Roy.  Mic.  8oc., 
1882,  p.  714)  stains  for  three  minutes  in  a  similar  solution 
without  the  alum,  washes,  and  stains  for  two  minutes  in  a  1 
per  cent,  aqueous  solution  of  methyl  green. 

The  liquid  of  Chenzinsky  has  been  given  (§  300).  It 
stains  nuclei  and  eosinophilous  granules. 

MEKKEI/S  carmine  and  indigo-carmine  stain  has  been  much 
recommended,  but  see  §  372. 

Fresh  (unfixed)  blood  may  be  treated  as  follows  (BizzozERO 
and  TORRE,  Archivio  per  le  Scienze  mediche,  vol.  iv,  No.  18, 
1880,  p.  390)  : — Dilute  a  drop  of  blood  with  0-75  per  cent, 
salt  solution  in  which  has  been  dissolved  a  little  methyl 
violet.  This  liquid  in  no  wise  affects  the  form  of  the  ele- 
ments, stains  intensely  the  nucleus  of  the  red  corpuscles,  and 
in  the  white  stains  the  nucleus  intensely,  and  the  protoplasm 
less  intensely.  May  be  used  for  the  study  of  bone  marrow 
and  spleen. 

For  the  staining  of  the  blood-plates  of  BIZZOZKRO,  this 
observer  (Archf  /.  path.  Anat.  u.  Phys.  ;  Zeit.  /.  wiss.  Mik., 
1884,  p.  389)  employs  a  0*02  per  cent,  solution  of  methyl 
violet  in  salt  solution,  or  a  1  :  3000  solution  of  gentian  violet. 

TOISON  (Journ.  Sci.  med.  de  Lille,  fev.,  1885  ;  Zeit.  /.  iviss. 
Mik.,  1885,  p.  398)  recommends  that  blood  be  mixed  with 
the  following  fluid  : 

Distilled  water      .  .  .      160  c.c. 

Glycerin  (neutral,  30°  Baume)        .       30    „ 
Pure  sulphate  of  sodium   .  .          8  grammes. 

Pure  chloride  of  sodium    .  .          1   gramme. 

Methyl  violet  5  B  .  0'25    „ 


SOME    OTHER   HISTOLOGICAL    METHODS.  451 

(The  methyl  violet  is  to  be  dissolved  in  the  glycerin  with 
one  half  of  the  water  added  to  it ;  the  two  salts  are  to  be 
dissolved  in  the  other  half  of  the  water,  and  the  two  solu- 
tions are  to  be  mixed  and  filtered.)  White  blood-cor- 
puscles stain  in  this  medium  in  five  or  ten  minutes;  the 
maximum  of  coloration  is  attained  in  from  twenty  to  thirty 
minutes.  White  blood- corpuscles,  violet ;  red  blood-cor- 
puscles, greenish. 

Ferrier's  liquid  is  said  to  have  a  sp.  gr.  similar  to  that  of 
liquor  sanguinis.  Fuchsin,  1  grm. ;  water,  150  c.c. ;  rectified 
spirit  50  c.c.  ;  dissolve,  and  add  glycerin,  200  c.c.  (from 
SQUIRE'S  Methods  and  Formula,  p.  39). 

For  UNNA'S  methods  for  staining  erythrocytes  in  alcohol-hardened 
tissues  (pathological  extravasations)  see  Ze.it.  /.  wiss.  Mik.,  xiii,  1896,  p. 
234. 

GiGLio-Tos  (Zeit.  f.  iviss.  Mik.,  xiv,  1897,  p.  359)  stains 
films  for  one  minute  in  a  warm  saturated  solution  of  methylen 
blue  B.X.,  and  examines  in  water. 

He  later  (ibid..,  xv,  1898,  p.  166)  mixes  fresh  blood  with 
a  saturated  solution  of  neutral  red  in  0*8  per  cent,  salt 
solution,  which  stains  hasmoglobigenous  granules  in  five  to 
ten  minutes. 

Neutral  red  is  also  recommended  as  an  intra  vitam  stain 
for  granules  of  leucocytes  by  EHRLICH  and  LAZARUS,  see 
§  296. 

ZIEMANN  (Zeit.  f.  iciss.  Mik.,  xv,  1899,  p.  456)  stains  films 
for  twenty  minutes  in  a  mixture  of  1  c.c.  of  1  per  cent, 
methylen  blue  and  5  c.c.  of  0*1  per  cent,  eosin  solution 
(mark  B.A.  or  A.G.,  Hochst). 

JENNKR'S  mixture,  see  §  807. 

PRINCE  (Micr.  Bull.,  xv,  1898,  p.  42  ;  Joarn.  Roy.  Mic.  Soc., 
1899,  p.  237 ;  Zeit.  f.  iciss.  Nik.,  xvi,  4,  1900,  p.  468)  adds 
to  twenty-four  parts  saturated  solution  of  toluidin  blue  one 
of  saturated  solution  of  Saurefuchsin  and  two  of  2  per  cent, 
eosin,  agitates  and  decants.  Films  are  stained  in  a  few 
seconds  in  the  fresh  solution,  or  in  a  few  minutes  after  it 
has  stood  for  some  weeks. 

It  goes  without  saying  that  the  Ehrlich-Biondi  mixture, 
(§  290),  and  Ehrlich's  triacid  and  acidophilous  mixtures, 
(§§  291  and  309)  may  be  found  valuable  in  many  haemato- 
logical  researches. 


452  CHAPTER    XXXII T. 

For  details  as  to  the  staining  reactions  of  the  granules  of  leucocytes,  see 
the  literature  quoted  in  previous  editions,  and  see  also  particularly  the- 
critique  in  FISCHER'S  "  Fixirung,  Fdrbung  und  Bau  des  Protoplasmas  " 
(Jena,  G.  Fischer,  1899),  the  outcome  of  which  is  that  the  supposed  speci- 
ficity of  the  reactions  is  a  mere  mare's  nest. 

809.  Demonstration  of  Blood-plates  of  Bizzozero  (KEMP,  Studies 
from  the  Biol.  Lab.  Johns  Hopkins  Univ.,  May,  1886,  iii,  No.  6; 
Nature,  1886,  p.  132). — A  somewhat  large  drop  of  blood  is 
placed  011  a  slide,  and  quickly  washed  with  a  small  stream  of 
normal  salt  solution.     The  blood-plates  are  not  washed  away, 
because  they  have  the  property  of  adhering  to  glass ;  and  on 
bringing  the  slide  under  the  microscope  they  will  be  seen  in 
large   numbers.      If   it  be  desired  to  make  permanent  pre- 
parations of  them,  they  should  first  be  fixed.      This  is  done 
by   putting    a    drop   of   osmic   acid    solution   on   the   finger 
before  pricking  it. 

For  BIZZOZERO'S  methods  for  the  numeration  of  these  elements  and  for 
the  study  of  their  regeneration,  see  his  paper  in  Festschr.  R.  Virchow 
gewidm.,  etc.,  1,  1891,  p.  459  ;  or  Zeit.f.  wiss.  Mik.,  ix,  2, 1892,  p.  229.  For 
his  stain  for  them  see  §  808. 

For  the  application  of  some  digestion  methods  to  the  study  of  blood-plates,, 
see  LILIENFELD,  Arch.  f.  Anat.  u.  Physiol.,  Physiol.  Abth.,  1892,  p.  115 ; 
or  Zeit.f.  wiss.  Mik.,  ix,  3,  1893,  p.  363. 

For  methods  for  obtaining  large  quantities  of  blood-plates,  see  DRTJEBIN 
Zeit.f.  wiss.  Mik.,  x,  4,  1893,  p.  493. 

For  BRODIE  and  RUSSELL'S  numeration  methods  see  Journ.  of  Physiol.* 
xxi,  1897,  p.  390;  Zeit.f.  wiss.  Mik.,  xiv,  1897,  p.  392. 

For  DETERMANN'S,  see  ibid.,  xvi,  1899.  p.  86. 

810.  WEIGERT'S  Fibrin  Stain    (Fortschr.  d.  Med.,  v,    1887, 
No.  8,  p.  228  ;  Zeit.  f.  wiss.  Mik.,  iv,  4,  1887,  p.  512).— Sec- 
tions (alcohol  -material)  are  stained  in  a  saturated  solution  of 
gentian    or    methyl   violet  in   anilin  water    (p.   203).      They 
are   brought   on   to   a   slide  and  mopped  up   with  blotting- 
paper,  and  a  little    Lugol's   solution  (§   88)   is  poured  on  to 
them.      After  this  has  been   allowed  to  act  for  a  sufficient 
time  they  are  differentiated  and  cleared  in  anilin  oil  without 
previous  dehydration  with  alcohol.      They  are  simply  mopped 
up  with  blotting-paper,  and  a  drop  of  anilin  is  poured  on  to 
them.      The  anilin  soon  becomes  dark,  and  is  then  changed 
for  fresh  once  or    twice.      When   this   has  been   done,   th& 
anilin  is  thoroughly  removed  by  means  of  xylol,  and  a  drop 


SOME    OTHER    H1STOLOGICAL    METHODS.  453 

of  balsam  and  a  cover  are  added.  This  stain  may  be  applied 
to  celloidin  sections  without  previous  removal  of  the  celloidin. 
See  also  the  modifications  of  this  method  by  KROMAYER  (§  656) 
and  BENECKE  (§  786). 


(Monatsschr.  prakt.  Dermat.,  xx,  1895,  p.  140  ;  Zeit.f.  wiss.  Mik., 
-xiii,  1896,  p.  229)  gives  a  modification  of  the  above  method,  and  also  one 
with  polychromatic  methylen  blue  and  iodide  of  potassium,  and  one  with 
fuchsin  and  tannin. 

WOLFF  (Zeit.  f.  wiss.  Mik.,  xv,  1899,  p.  310)  makes  up  the  stain  with 
two  vols.  saturated  solution  of  carbonate  of  lithia  to  one  of  alcoholic 
gentian  or  fuchsin  ;  other  details  loc.  cit.,  or  Journ.  Roy.  Mic.  Soc.,  1899, 
p.  234. 


Glands. 

811.  Mucin. — HOYER  (Arch.  /.  mik.  Anat.,  xxxvi,  1890, 
p.  310;  Zeit.f.  u-iss.Mik.,  viii,  1,  1891,  p.  67)  has  the  follow- 
ing conclusions  : 

The  "  mucin  "  of  mucus  cells  and  goblet  cells  stains  with 
basic  tar  colours  and  with  alum  haematoxylin,  but  not  with 
acid  tar  colours  (see  §  262).  He  obtained  his  best  results 
by  means  of  thionin,  and  good  ones  with  toluidin  blue,  both 
of  these  giving  a  metachromatic  stain — tissues  blue,  mucin 
reddish — and  also  with  methylen  blue  (which  is  particularly 
useful  from  its  power  of  bringing  out  the  merest  traces  of 
mucin),  safranin,  etc. 

All  of  these  colours  may  be  used  in  the  same  way.  Speci- 
mens should  be  fixed  for  two  to  eight  hours  in  5  per  cent, 
sublimate  solution,  imbedded  in  paraffin,  cut,  and  the  sec- 
tions stained  for  five  to  fifteen  minutes  in  a  very  dilute 
aqueous  solution  of  the  dye  (two  drops  of  saturated  solution 
to  5  c.c.  of  water). 

It  is  theoretically  interesting  to  observe  that  hyaline  car- 
tilage, the  jelly  of  Wharton,  and  the  Mastzellen  of  Ehrlich 
give  the  same  reactions  with  basic  dyes  as  mucin  does,  even 
their  metachromatic  reactions  being  identical. 

These  conclusions  had  already  been  in  part  formulated  by 
SUSSDORP  (Deutsche  Zeit.f.  Thiermed.,  xiv,  pp.  345,  349;  see 
Zeit.f.  iciss.  Mik.,  vi,  2,  1889,  p.  205). 

See  also  the  series  of  papers  by  BIZZOZERO,  "  Sulle  ghian- 
dole  tubulari  del  tubo  gastro-enterico,"  etc.,  in  the  Atti  R. 


454  CHAPTER  XXXIII. 

Accad.  di  Sci.  di  Torino,  1889  to  1892  ;  reports  in  Zeit.  /. 
wiss.  MiJc.}  vii,  1,  1890,  p.  61 ;  and  ix,  2,  1892,  p.  219  ;  also 
UNNA,  ibid.,  xiii,  1896,  p.  42. 

As  regards  the  safranin  reaction,  it  is  well  to  note  that  it 
is  not  obtained  with  all  brands  of  the  dye  ;  that  of  Bind- 
schedler  and  Busch,  in  Bale,  gives  it,  whilst  safranin  0  of 
Grriibler  does  not.  UNNA  employs  chiefly  his  polychromatic 
methylen  blue  (§§  321,  794). 

The  subject  has  been  carefully  investigated  by  PAUL 
MAYER  (Mitth.  Zool  Stat.  Neapel,  xii,  2,  1896,  p.  303;  Zeit. 
f.  wiss.  Mils.,  xiii,  1896,  p.  38).  As  regards  the  haematein 
reaction,  he  finds — 

1.  If  the  staining   solution   contain  free   acid,  or  a  rela- 
tively large   amount    of   alum,  then   as   a   general  rule  the 
secretion  of  mucus  gland  cells  does  not  stain  in  it. 

2.  If  it  contain  a  relatively  small  proportion  of  alum,  but 
a  large  proportion  of  haematein,  then  it  stains  many  sorts  of 
mucus,  and  at  the  same  time  stains  chromatin  strongly. 

He  also  finds  that  mixtures  of  a  relatively  large  amount 
of  methyl  violet  and  a  relatively  small  amount  of  methylen 
blue  with  a  little  acetic  acid  stain  nuclei  blue,  mucus  red 
(p.  314).  Unna's  methylen  blue  stains  well;  the  red  stain 
of  thionin  keeps  badly,  that  of  toluidin  blue  somewhat 
better.  A  sharp  stain  that  keeps  well  in  balsam  is  got  by 
staining  for  several  hours  in  a  concentrated  solution  of 
safranin  in  30  per  cent,  alcohol  with  a  very  little  hydro- 
chloric acid.  Mucus  from  different  sources  reacts  very 
differently,  and  there  are  certainly  several  sorts  of  mucin. 
At  the  same  time,  thionin,  safranin,  and  other  colours,, 
quoted  by  HOYER  as  specific  stains  for  mucin,  stain  in  the 
same  way  substances  that  are  not  mucin,  e.  g.  Corpora 
amylacea,  albumin,  gum  arabic.  He  gives  the  following  two 
formulas  for  mixtures  that  stain  exclusively  mucus  (the 
distinction  between  mucin  and  mucigen  is  not  taken  in  his. 
paper) . 

812.  MAYER'S  Mucicarmine  (op.  cit.,  last  §). — One  gramme 
of  carmine  is  rubbed  up  in  a  capsule  with  0'5  gramme  of 
aluminium  chloride  (must  be  dry,  not  damp  and  yellow), 
and  2  c.c.  of  distilled  water.  The  capsule  is  heated  over  a 
small  flame  for  two  minutes,  until  the  originally  light -red. 


SOME    OTHEK    H1STOLOGICAL    METHODS.  455 

mixture  has  become  quite  dark.  Stir  thoroughly.  The 
liquid  having  become  thick,  add  a  little  50  per  cent,  alcohol, 
in  which  the  warm  mass  ought  to  dissolve  easily,  and  rinse 
the  whole  with  more  alcohol  into  a  bottle.  Make  up  to  100 
c.c.  with  50  per  cent,  alcohol,  let  it  stand  for  at  least 
twenty-four  hours,  and  filter.  This  gives  a  stock  solution, 
which  is  as  a  rule  to  be  diluted  for  use  tenfold  with  distilled 
or  tap  water.  Exceptionally  it  may  be  diluted  instead  five 
or  ten  fold  with  alcohol  of  50  per  cent,  or  70  per  cent. 
The  stock  solution  may  be  obtained  from  Griibler  and  Co, 
Mucicarmine  stains  in  sections  or  thin  membranes  mucus 
only.  Nuclei  may  be  stained  before  with  haemalum. 

813.  MAYER'S  Muchaematein  (ibid.). — Haematein  0'2  gramme, 
aluminium  chloride  O'l  gramme,  glycerin  40  c.c.,  water  60  c.c. 
Eub  up  the  haematein  in  a  mortar  with  a  few  drops  of  the 
glycerin,  then  add  the  other  ingredients.      If  it  be  desired 
to   avoid   employing   a  watery  liquid,   an  alcoholic   solution 
may  be  made  in  the  same  way  by  dissolving  the  haematein 
and  aluminium  chloride  in   100  c.c.  of  70  per  cent,  alcohol, 
with    or  without  the   addition  of  two  drops  of   nitric  acid. 
This    is    a    nearly    pure    mucus    xtain    for    sections   or    thin 
membranes.      Nuclei   may  be    stained   before  with   paracar- 
mine. 

If  the  mucus  swells  much  (as  in  Fishes)  the  alcoholic  solutions  of  inuci- 
carmine  or  muchsematein  are  indicated,  and  watery  fixatives  should  be 
avoided  as  much  as  possible, 

814.  Mucicarminic    Acid    (RAWITZ,  Anat.  Anz.,    xv,   1899, 
p.  439). — 1  gramme  of  carminic  acid,  2  of  aluminium  chloride, 
and    100    c.c.    of   50   per   cent,    alcohol    are   dissolved    and 
evaporated  to  dryness  on  a  sand-bath  and  the  residue  taken 
up   with    100   c.c.    of  50  per  cent,  alcohol.      Use  as  muci- 
carmine. 

815.  Neutral  Red.— KULTSCHIZKY  (Arch.  mik.  Anat.,  xlix,  1897,  p.  8) 
fixes  in  his  mixture  (end  of  §59),  and  stains  sections  either  in  safranin  with 
2  percent,  acetic  acid,  or  in  a  similar  solution  of  neutral  red  (two. to  three 
days,  washing  out  with  alcohol). 

816.  Goblet  Cells. — So  far  as  these  contain  mucin  they  give 
the  reactions  above  described,  see  FLEMMING,  Zeit.  f.  wiss. 


156  CHAPTER  XXXIII. 

Mik.,  1885,  p.  519;  PAULSEN,  ibid.,  p.  520;  PANETH,  Arch, 
f.  mik.  Anat.,  xxxi,  1888,  p.  113  et  seq.  ;  and  LIST,  ibid., 
xxvii,  1886,  p.  481. 

RANVIEE  (Comptes  rend.,  1887,  3,  p.  145  ;  Zeit.f.  wiss.  Mik.,  v,2, 1888, 
p.  233)  treats  the  pharyngeal  mucosa  of  the  frog  first  for  ten  or  twelve 
hours  with  vapour  of  osmium,  and  then  for  three  minutes  with  vapours  of 
perruthenic  acid  (Ru04)  and  obtains  the  mucigen  in  the  goblet  cells  stained 
black. 

817.  Salivary  Glands. — SOLGER    (Unters.    z.    Naturlehre    d. 
Menschen,  xv,  5  and  6,  pp.  2 — 15;   Festschr.  f.    Gegenbaur, 
ii,  1896,  p.  211;  Zeit.  f.   wiss.  Mik.,   xii,   3,    1896,  p.  374) 
demonstrates  the  granules  in  serous  cells  and  half-moons  of 
the   submaxillary   gland  by   means   of   formaldehyde.      The 
gland  is  hardened  for  two  days  or  more  in  a   10  per  cent, 
solution  of  formol,  and  may  then   either  be   sectioned   and 
examined  in  the  wet  way  or  imbedded  in  paraffin,  and  the 
sections  stained  with  hsematoxylin  of  Delafield  or  of  Ehrlich, 
the  granules  taking  the  stain. 

KKAUSE  (Arch.  f.  mik.  An  at.,  xlv,  1895,  p.  94)  stains 
sections  either  with  Heidenhain's  iron  hsematoxylin  or  with 
Ehrlich-Biondi  mixture  or  thionin.  See  also  KRAUSE,  ibid., 
xlix,  1897,  p.  709. 

818.  Glands  of  Larynx  and  Trachea. — FUCHS-WOLFRING  (Arch, 
mik.  Anat.,  Hi,  1898,  p.  735  ;  Zeit.  f.  wiss.  Mik.,  xv,  2,  1898, 
p.  232)  found  mucicarmine  the  best  reagent  for  mucus.      It 
is  advantageous  to  employ  it  with  haemalum   (§  812).      He 
also  used  a  light  stain  with  Delafield's  hsematoxylin  followed 
by    Congo    red    (demonstrates    the    secretory    capillaries    of 
serous  glands). 

819.  Gastric   Glands. — KOLSTER    (Zeit.  f.    wiss.    Mik.,   xii, 
1895,  p.  314)  differentiates  the  two  kinds  of  cells  in  stomach 
glands  by  over-staining  with  heematoxylin,  washing  out  with 
alcohol  containing   1  per  cent,  of  HC1,  blueing  with  alcohol 
containing    1    per    cent,    of    ammonia,    and,    after    washing, 
staining   for    one    to    five    minutes    in    a    weak    solution    of 
Saurefuchsiii.      Peptic  cells  blue,  parietal  cells  red.      Osmic 
material  cannot  be  employed. 

See  also  OPPEL,  Lehrb.  Vergl.  Anat.  Wirbelthiere,  1,  Der  Magen,  Jena,. 
1896. 


SOME    OTHER    HISTOLOGICAL    METHODS.  457 

820.  Liver. — BRAUS    (Denkschr.   Med.   Nat.    Get.    Jena,   v, 
1896,  p.  307)   demonstrates  the  bile  capillaries  by  the  rapid 
method  of  GOLGI,  hardening  in  a  mixture  of  one  part  formol 
with   three  parts   liquid   of   Miiller  or  ^  per  cent,   chromic 
acid.       He   also   stains  with   Bordeaux   B  and  iron  haema- 
toxylin,   or  with  Ehrlich-Biondi   mixture,   after   fixing  in  a 
mixture  of  one  part  formol  to  three  of  7^  per  cent,  sublimate 
solution. 

HOLM  (Zool.  Jahrb.,  Abth.  Morph.,  x,  1897,  p.  283)  fixes  the  extremely 
fatty  liver  of  Acanthias  in  a  mixture  of  5  parts  alcohol  and  1  of  chloroform, 
and  imbeds  in  paraffin. 

OPPEL  (Anat.  Anz.,  v,  1890,  p.  144;  vi,  1891,  p.  168)  puts 
pieces  of  liver  or  spleen  (alcohol  material)  for  twenty-four 
hours  into  a  solution  of  neutral  chromate  of  potash  (^  to 
10  per  cent.),  rinses  with  a  very  weak  solution  of  silver 
nitrate,  puts  them  for  twenty-four  hours  into  a  £  per  cent, 
solution  of  silver  nitrate,  washes,  dehydrates  and  cuts 
without  imbedding.  The  lattice  fibres  are  only  stained  near 
the  surface,  so  that  tangential  sections  must  be  made. 

See  also  KANVIEB,  "  Les  membranes  muqiieuses  et  le  syst.  glandulaire" 
Journ.  de  Microgr.,  ix,  x,  1885-6  ;  IGACUSCHI,  in  Arch.  f.  path.  Anat., 
xcvii,  p.  142,  or  Zeit.f.  vriss.  Mik.,  1885,  p.  243  (gold  process  for  study  of 
fibrous  networks) ;  KUPFFEH,  Sitzb.  Ges.  f.  Morph.,  etc.,  Miinchen,  Juli, 
1889,  or  Zeit.  /.  wise.  Mik.,  vi,  4,  1889,  p.  506  (hsematoxylin  stain  for 
demonstration  of  ultimate  bile-ducts,  and  application  of  Golgi's  silver  bi- 
chromate method  to  the  same  object  and  to  the  study  of  fibrous  networks)  ; 
KBAUSE,  Arch.  mik.  Anat,,  xlii,  1893,  p.  57. 

821.  Spleen. — For  the  lattice  fibres,  see  OPPEL,  last  §. 
KULTSCHITZKY  (Arch.  mik.  Anat.,  xlvi,  1895,  p.  675)  studies 

the  musculature  in  sections  (of  material  from  liquid  of  Miiller) 
stained  for  a  day  or  more  in  a  solution  of  "  lakmoid  "  in  ether 
and  mounted  in  balsam. 

For  elastic  fibres  he  puts  sections  for  half  an  hour  or  a 
day  into  a  mixture  of  800  parts  96  per  cent,  alcohol,  40  parts 
1  per  cent,  solution  of  carbonate  of  potash,  2  parts  Magdala 
red,  and  1  part  methylen  blue. 

For  the  blood-vessels  he  puts  sections  of  Miiller  material 
for  a  few  minutes  into  a  solution  of  one  or  two  parts  of 
Saurerubin  in  400  parts  of  3  per  cent,  acetic  acid,  washes 
out  in  2  per  cent,  acetic  acid,  and  after- stains  in  a  similar 


458  CHAPTER    XXX II I. 

solution   of    helianthin    or   Wasserblau   until    the    red   only 
remains  in  the  erythrocytes. 

See  also  WHITING,  Trans.  Roy.  Soc.,  Edinburgh,  xxxviii,  1896,  p.  311. 

822.  Kidney.— SAUER  (Arch.  mik.  Anat.,  xlvi,  1895,  p.  110) 
has  a  thorough  discussion  of  the  methods  for  the  study  of 
the  renal  epithelium.  He  finds  the  best  fixative  is  Carnoy's 
acetic  alcohol  with  chloroform,  §  88  (three  to  five  hours, 
washing  out  with  absolute  alcohol) .  A  mixture  of  nine  parts 
alcohol  with  one  of  nitric  acid  is  also  good,  as  is  liquid  of 
Perenyi.  He  stains  with  iron  ha3matoxylin,  and  after- stains 
in  a  very  weak  solution  of  Saurerubin  in  90  per  cent,  alcohol, 
which  stains  the  ciliary  plateau.  He  macerates  with  iodised 
serum  or  one  third  alcohol,  staining  afterwards  with  dahlia. 


CHAPTER  XXXIV. 

SOME   METHODS    FOB    LOWER   ANIMALS. 

823.  Introduction. — The  following  methods  are  all  of  them 
such  as  give  results  applicable  to  histological  study,  and  no 
account  has  been  taken  of  such  methods  as  are  merely  useful 
for  the  preparation  of  organisms  for  museum  specimens  or 
for  coarse  dissection. 

On  p.  74  will  be  found  a  note  of  warning  as  to  the  employment  of  the 
now  fashionable  formaldehyde  as  a  preservative. 

A  valuable  paper  giving  an  account  of  a  number  of  the  processes  employed 
in  the  Naples  Zoological  Station  for  the  preservation  of  marine  animals  has 
been  published  by  SALTATOBE  Lo  BIANCO  in  Mitth.  Zool.  Stat.  Neapel,  ix, 
1890,  p.  435.  References  to  the  work  of  S.  Lo  BIAXCO  in  the  remainder  of 
this  chapter  are  to  that  paper.  An  abstract  of  it  is  contained  in  Amer. 
Natural,  xxiv,  1890,  p.  856,  and  Journ.  Roy.  Mic.  Soc.,  1891,  p.  133,  and 
a  very  full  account  in  Zeit.f.  wiss.  Mik.,  viii,  1,  1891,  p.  54. 

Tunica  ta. 

824.  Fixation  of  Tunicata. — A  method  of    SALVATOKE   Lo 
BJAXCO  for  killing  simple  Ascidians  in  an  extended  state  has 
been  given  above,  §  22.      In  the   paper   quoted   above  this 
plan  is  recommended  for  Ciona,  Ascidia,  and  Rhopalea.      But 
many  other  forms,  such  as  Clavellina,  Perophora,  Phallusia, 
Molgula,  Cynthia,  etc.,  should  first   be   narcotised  by  treat- 
ment for  from  three  to   twelve  hours  with   chloral   hydrate 
(1  :  1000  in  sea  water),  then  killed  in  a  mixture  containing 
chromic  acid  of    1  per  cent.  10  parts,  acetic  acid  10U  parts, 
and  finally  hardened  in  1  per  cent,  chromic  acid. 

The  compound  Ascidians  with  contractile  zooids  are  diffi- 
cult to  manage  if  one  does  not  go  the  right  way  to  work. 
The  best  process  known  to  me  is  the  following  (due  to  VAN 


460  CHAPTER   XXXI V. 

BENEDEN,  kindly  communicated  to  me  by  Dr.  C.  Maurice). 
Place  the  corms  in  clean  sea  water,  and  leave  them  alone  for 
a  few  hours,  in  order  that  the  zooids  may  become  fully 
extended,  then  plunge  them  suddenly  into  glacial  acetic  acid. 
Leave  them  there  for  two,  four,  or  six  minutes,  according  to 
the  size  of  the  corms  (which  are  best  taken  of  as  small  a  size 
as  possible).  Take  them  out  of  the  acid  with  your  fingers 
(or  at  all  events  not  with  steel  instruments,  which  would 
blacken  the  tissues)  and  bring  them  into  50  per  cent,  alcohol. 
Wash  them  thoroughly  in  that,  and  then  bring  them  in  the 
usual  way  through  successively  stronger  alcohols. 

I  strongly  recommend  this  process,  which  gives  admirably 
preserved  preparations  quite  free  from  any  opacity  either  in 
the  tissues  or  the  tunic.  The  acid  will  not  hurt  the  fingers 
if  they  be  washed  immediately. 

S.  Lo  BIANCO  recommends  for  this  group  the  chloral 
hydrate  process,  followed  by  fixation  with  sublimate  or 
chromo-acetic  acid. 

CATJLLERY  (Bull.  Sc.  France  Belg.,  xxvii,  1895,  p.  5)  first  stupefies  the 
animals  with  cocaine  (LAHILLE,  a  few  drops  of  5  per  cent,  solution  to  30  c.c. 
of  sea  water),  then  fixes  in  liquid  of  Flemming  or  acetic  acid. 

Most  small  pelagic  Tunicates  are  very  easily  fixed  with 
osmic  acid  or  acid  sublimate  solution. 

I  have  found  the  acetic  acid  process  very  good  for  Pyro- 
soma.  Lo  BIANCO  puts  them  for  a  quarter  of  an  hour  into 
50  per  cent,  alcohol  containing  5  per  cent,  of  hydrochloric 
acid,  then  into  successive  alcohols,  beginning  with  60  per 
cent.  He  kills  the  hard  forms  of  Salpa  with  acetic  acid  of 
10  per  cent.,  the  semi- hard  ones  with  1  per  cent,  chromic 
acid  containing  5  per  cent,  acetic  acid,  the  soft  ones  with  1 
per  cent,  chromic  acid  containing  ^  per  cent,  osmic  acid, 
Doliolidae  with  sublimate,  or  the  above  osmic  mixture,  or 
a  mixture  of  10  parts  10  per  cent,  solution  of  sulphate 
of  copper  with  one  part  concentrated  sublimate  solution. 


Molluscoida. 

825.  Bryozoa. — For  some  methods  of  killing  and  fixing  see 
§§   Hj  18,  and  19.      S.  Lo   BIANCO  employs  for  Pedicellina 


SOME    METHODS    FOR    LOWtiR  ANIMALS.  461 

and  Loxosoma  the  chloral  hydrate  method,  fixing  with  sub- 
limate. For  Flustra,  Cellepora,  Bugula,  Zoobothrium,  he 
employs  the  alcohol  method  of  EISIG,  §  16.  For  Cristatella 
see  §§  17,  18. 

CONSKR  (Trans.  Amer.  Mic.  Soc.,  xvii,  1896,  p.  310)  kills 
the  fresh-water  forms  with  cocaine,  puts  them  for  an  hour 
into  1  per  cent,  chromic  acid,  and  passes  through  water  into 
alcohol,  etc. 

826.  Brachiopoda.— Lo  BIANCO  kills  small  animals  in  70  per 
cent,  alcohol,  larger  ones  in  the  same,  but  after  first  nar- 
cotising with  alcohol  and  sea  water. 

BLOCHMANN  (Untersuch.fein.  Ban  Brachiopoden,  Jena,  1892, 
p.  5)  fixes  principally  with  sublimate,  macerates  by  the 
HERTWIGS'  method,  §  538,  decalcifies  with  1  per  cent,  chromic 
acid  (for  thick  shells  add  a  little  hydrochloric  or  nitric  acid), 
or  with  nitric  acid  in  alcohol  of  50  to  70  per  cent.,  and  im- 
beds in  paraffin  or  celloidin,  the  latter  giving  the  less 
shrinkage.  For  injections,  Berlin  blue,  or  2  per  cent,  blue 
or  red  gelatin. 

EKMAN  (Zeit.  u-iss.  Zool.,  Ixii,  1896,  p.  172)  fixes  the 
peduncle  chiefly  with  liquid  of  Flemming,  and  cuts  chiefly 
with  the  free  hand,  in  liver,  seldom  in  paraffin. 


Mollusca. 

827.  Fixation  of  Mollusca. — Two  groups  at  least  amongst 
the  Mollusca  offer  considerable  difficulties  in  the  way  of  fixa- 
tion— Lamellibranchiata  and  Gastropoda. 

Lo  BIANCO  narcotises  Lamellibranchs  for  six  to  ten  hours 
or  more  with  alcohol,  §  16,  and  then  kills  them. 

CARAZZI  (Mitth.  Zool.  Stat.  Neapel,  xii,  1896,  p.  388)  also 
employs  the  alcohol  method,  but  warms  the  vessel  containing 
the  animals  to  about  25°  C.,  and  thus  obtains  total  narcosis 
in  twenty-four  hours.  For  the  study  of  the  branchiae  he 
then  excises  all  four  lamellae,  fixes  them  in  a  sublimate 
mixture  similar  to  that  of  Gilson,  §  70,  for  one  or  two  hours 
(or  the  entire  body  of  the  animal  may  be  excised  and  fixed 
for  four  to  six  hours),  then  passes  through  iodine-alcohol  and 
absolute  alcohol,  removes  the  two  outer  lamellae,  and  imbeds 


462  CHAPTER    XXXIV. 

the  two  inner  ones,  which  have  suffered  less  from  the  opera- 
tions. 

See  also  §  20. 

The  methods  recommended  for  Lamellibranchiata  some- 
times give  good  results  with  Gastropoda. 

S.  Lo  BIANCO  advises  that  Prosobranchiata,  and,  amongst 
the  Heteropoda,  Atlantidae,  be  narcotised  with  70  per  cent, 
alcohol,  §  16.  Opisthobranchiata  ought  not  to  give  much 
trouble,  and  I  recommend  sudden  killing  with  liquid  of 
Perenyi,  or  the  acetic  method,  §  824.  Aplysia  may  first  be 
narcotised  by  subcutaneous  injection  of  about  1  c.c.  of  a  5 
to  10  per  cent,  solution  of  hydrochlorate  of  cocaine  (ROBERT, 
Bull.  Scient.  de  la  France,  etc.,  1890,  p.  449  ;  Zeit.f.  wiss.  Nik., 
ix,  2,  1892,  p.  216),  or  (SCHONLEIN,  Zeit.  Biol.,  xxx,  1893, 
p.  187)  1  c.c.  of  4  per  cent,  solution  of  Pelletierin.  For 
Lo  BIANCO'S  various  methods  see  the  original,  p.  467. 

For  Pteropoda  in  general,  liquid  of  Perenyi.  Creseia  is  a 
difficult  form.  S.  Lo  BIANCO  advises  the  alcohol  method, 
§  16.  For  the  Gymnosomata  he  narcotises  with  0*1  per  cent, 
chloral  hydrate. 

Note  the  hydroxylamin  method  of  HOFER,  §  20. 

For  preservation  it  may  be  noted  that  for  Heteropoda  and  Pteropoda, 
formaldehyde  (preceded  by  due  fixation  in  a  chromic  or  sublimate  solution) 
is  an  admirable  medium,  so  far  at  least  as  macroscopic  appearances  are  con- 
cerned, and  for  this  purpose  superior  to  alcohol. 

For  terrestrial  Gastropods  see  the  Asphyxiation  method, 
§  23.  See  for  removal  of  mucus,  MAECHI,  Arch.  mik.  Anat., 
1867,  p.  204. 


828.  Eyes  of  Gastropoda  (FLEMMING,  Arch.  f.  mik.  Anat., 
1870,  p.  441). — The  difficulty  here  is  to  obtain  the  excision 
of  an  exserted  eye.  It  is  impossible  to  sever  the  exserted 
peduncle  in  a  living  animal  without  its  retracting,  at  least 
partially,  before  the  cut  is  completed.  Never  mind  that ;  make 
a  rapid  cut  at  the  base,  and  throw  the  organ  into  very  dilute 
chromic  acid,  or  4  per  cent,  bichromate ;  after  a  short  time 
it  will  evaginate,  and  remain  as  completely  erect  as  if  alive. 
Harden  in  1  per  cent,  osmic  acid,  in  alcohol,  or  in  bichromate. 

CARRIERS    (Zool.   Anz.,  1886,  p.    221)    removes    the    eye, 


SOME    METHODS   FOB   LOWER   ANIMALS.  463 

together  with  a  portion  of  the  tentacle,  and  fixes  by  exposing 
it  for  some  minutes  to  vapour  of  osmic  acid.  He  depigments 
sections  by  very  careful  treatment  with  very  dilute  eau  de 
Javelle. 

829.  Eyes  of  Cephalopoda  and  Heteropoda  (GRENACHER,  Abh. 
naturf.  Ge*.  Halle-a.-S.,  Bd.  xvi ;  Zeit.  f.  iciss.  Hik.,  1885, 
p.  244). — Fix  (Cephalopod  eyes)  in  picro-sulphuric  acid,  or 
in  a  saturated  solution  of  corrosive  sublimate  in  picro-sul- 
phuric acid  (this  mixture  is  especially  useful  for  Octopus, 
Eledone,  and  Sepia,  but  does  not  succeed  with  the  pelagic 
forms,  such  as  Loligo,  Ommatostrephes,  and  Rossia).  Depig- 
ment  the  specimens  with  hydrochloric  acid  (in  preference  to 
nitric  acid).  The  mixture  §  581  may  also  be  used.  The 
operation  of  depigmentation  may  be  combined  with  that  of 
staining ;  if  you  stain  with  borax-carmine  and  wash  out  in 
the  last-mentioned  mixture  the  pigment  will  be  found  to  be 
removed  quicker  than  the  stain  is  washed  out.  The  operation 
may  be  carried  out  on  sections,  but  it  is  better  to  use  portions 
of  retina  of  2  to  5  mm.  in  thickness.  Grenacher  mounted 
his  preparations  in  castor  oil,  see  §  447. 

Similar  methods  are  recommended  by  the  same  author  for 
the  eyes  of  Heteropoda  (see  Abh.  naturf.  Ges.  Halle-a.-S., 
1886;  Zeit.  f.  wiss.  Mik.,  1886,  p.  243). 

LENHOSSEK  (Zeit.  iinss.  Zool.,  Iviii,  1894,  p.  636;  Arch.  mik.  Anat., 
xlvii,  1896,  p.  45)  applies  the  method  of  GOLOI  to  the  eyes  of  Cephalopods. 

Similarly  KOPSCH  (Anat.  Anz.,  xi,  1895,  p.  362),  but  using  formol  instead 
of  the  osmic  acid. 

830.  Eyes  of  Lamellibranchiata.— See  PATTEN,  Mitth.  Zool.  Stat. 
Neapel,  vi,  1886,  p.  733,  and  RAWITZ,  Jena,  Zeit.  Naturw.,  xxii,  1888, 
p.  115,  and  xxiv,  1890,  p.  579  (bleaches  with  caustic  soda) ;  see  §  584. 

831.  Central  Nervous  System  of  Pulmonata. — B.  de  NABIAS 
(Act.  Soc.  Linn.  Bordeaux,  1894;  Rech.  Hist,  centres  nerveux 
de*  Gasteropodes,  1894,  p.  23)  opens  the  animals  and  fixes 
the  ganglia  for  one  hour  in  a  mixture  of  6  parts  glacial  acetic 
acid  to  100  of  90  per  cent,  alcohol,  or  for  fifteen  to  twenty 
minutes  in  5  per  cent,  sublimate  with  5  per  cent,  acetic  acid. 
He  stains  in  bulk,  with  Renaut's  haematoxylic  eosin,  §  389, 
or  R.  Heidenhain's  hsematoxylin,  §  252,  or  a  copper  haema- 


464  CHAPTER    XXXIV. 

toxylin  of  Viallanes,  and  imbeds  in  paraffin.  He  also  stains 
by  the  rapid  method  of  GOLGI,  imbedding,  however,  the 
ganglia  in  celloidin  directly  after  the  hardening  in  osmic 
acid  and  bichromate,  and  treating  the  sections  with  the 
silver  (p.  34).  He  stains  with  methylen  blue  by  treating 
the  ganglia  in  situ  for  twelve  to  twenty-four  hours  with  a 
1  per  cent,  solution. 

832.  Shell. — Sections    of    non-decalcified   shell   are    easily 
obtained  by  the  usual  methods  of  grinding,  or,  which  is  often 
a  better  plan,  by  the  methods  of  v.  Koch  or  Ehrenbaum, 
§§   172,  173.      MOSELEY   (Quart.  Journ.  Mic.   Sci.    [2],  xxv, 
1885,  p.  40)   decalcifies  with  nitric  acid  of  3  to  4  per  cent, 
and  then  makes  sections. 

833.  Injection  of  Acephala  (FLEMMING,  Arch.  f.  mik.  Anat., 
1878,  p.  252). — To  kill  the  animals  freeze  them  in  a  salt-and- 
ice  mixture,  and  throw  them  for  half  an  hour  into  lukewarm 
water.      They  will  be  found  dead,  and  in  a  fit   state  for  in- 
jection.     Chloroform  and  ether  are  useless  (but  see  §  20). 
The  injection-pipe  may  be  tied  in  the  heart ;  but  when   this 
has  been  accomplished  there  remains  the  problem  of  occlud- 
ing cut  vessels  that  it  is  impossible  to  tie.      To  this  end,  after 
the  pipe  has  been  tied,  the  entire  animal  is  filled  and  covered 
up  with  plaster  of  Paris.     As  soon  as  the  plaster  has  hardened 
the  injection  may  be  proceeded  with. 

See  also  DEWITZ,  Anleit.  zur   anfert.    zootom.     Prap.,   Berlin,  1886, 
p.  44  (Anodonta)  and  p.  52  (Helix}. 

834.  Maceration     Methods     for     Epithelium.  —  BNGELMANN 
(P finger's  Arch.,  xxiii,  1880,  p.  505)   macerates  the  intestine 
of  Cyclas  in  osmic  acid  of  0'2  per  cent,  (after  having  warmed 
the  animal  for  a  short  time  to  45°  to  50°  0.),  or  in  concen- 
trated boracic  acid  solution. 

The  Intr a- cellular  Processes  of  the  Cilia. — The  entire  intra- 
cellular  fibre  apparatus  may  be  isolated  by  teasing  fresh 
epithelium  from  the  intestine  of  a  Lamellibranch  (e.  g. 
Anodonta)  in  either  bichromate  of  potash  of  4  per  cent.,  or 
salt  solution  of  10  per  cent.  To  get  good  views  of  the 
apparatus  in  situ  in  the  body  of  the  cell,  macerate  for  not 


SOME    METHODS    FOB    LOWER    ANIMALS.  465 

more  than  an  hour  in  concentrated  solution  of  boracic  or 
salicylic  acid.  Very  dilute  osmic  acid  (e.g.  O'l  per  cent.) 
gives  also  good  results.  The  "  lateral  cells  "  of  the  gills  are 
best  treated  with  strong  boracic  acid  solution  (five  parts  cold 
saturated  aqueous  solution  to  one  part  water). 

BELA  HALLER'S  Mixture,  see  §  539. 

BROCK'S  Medium,  §  535. 

MOBIUS'S  Media,  §  536 ;  the  second  of  these  is  much 
recommended  by  DROST  (Morphol.  Jahrb.,  xii,  2,  1866,  p.  163) 
for  Cardium  and  Mya. 

See  also  PATTEN  (Mitth.  Zool.  Stat.  Neapel,  vi,  4,  1886, 
p.  736).  Sulphuric  acid,  40  drops  to  50  grammes  of  water, 
is  here  recommended  as  a  valuable  macerating  and  preserva- 
tive agent.  Entire  molluscs,  without  the  shell,  may  be  kept 
in  it  for  months. 

BERNARD  (Ann.  Sci.  Nat.,  ix,  1890,  p.  191)  macerates  the 
mantle  of  Prosobranchs  in  a  mixture  of  one  part  each  of 
glycerin  and  acetic  acid,  two  parts  each  of  90  per  cent, 
alcohol  and  O'l  per  cent,  chromic  acid,  and  forty  parts  water, 
which  acts  in  from  a  quarter  of  an  hour  to  three  hours.  He 
also  (pp.  102,  306)  uses  a  weak  solution  of  chloride  of 
ruthenium,  especially  for  nerve  tracts,  mucus  cells  and  cilia. 
Alcohol  material  may  be  macerated  in  a  mixture  of  one  part 
glycerin,  two  of  acetic  acid,  and  forty  of  water. 


Arthropoda. 

835.  General  Methods  for  Arthropoda. — As  general  methods 
for  the  study  of  chitinous  structures,  the  methods  worked 
out  by  Paul  Mayer  (see  §§  7  and  9,  and  also  90,  229,  and 
230)  are  excellent.  It  is  at  all  events  absolutely  necessary 
that  all  processes  of  fixation,  washing,  and  staining  should  be 
done  with  fluids  possessing  great  penetrating  power.  Hence 
picric  acid  combinations  should  in  general  be  used  for  fixing, 
and  alcoholic  fluids  for  washing  and  staining.  Concentrated 
picro-sulphuric  acid  (or  picro-nitric)  is  the  most  generally 
useful  fixative,  and  70  per  cent,  alcohol  is  the  most  useful 
strength  for  washing  out. 

Alcoholic  picro-sulphuric  acid  may  be  indicated  for  fixing 

in  some  cases. 

30 


466  CHAPTER   XXX IV. 

Some  forms  are  very  satisfactorily  fixed  with  sublimate. 
Such  are  the  Copepoda  and  the  larvae  of  Decapoda.  It  is 
sometimes  indicated  to  use  the  sublimate  in  alcoholic  solution. 
Some  Copepoda,  however  (Copilia,  Sapphirina),  are  better 
preserved  by  means  of  weak  osmic  acid,  and  so  are  the  Ostra- 
coda.  In  many  cases  the  osmic  acid  will  produce  a  sufficient 
differentiation  of  the  tissues,  so  that  further  staining  may 
be  dispensed  with  ;  Copilia  and  Phyllosoma  are  examples  of 
forms  that  may  be  prepared  in  this  simple  manner.  The 
pyrogallic  process  (§  361)  may  often  prove  helpful  in  the 
study  of  such  forms. 

For  Ostracoda,  MULLER  (Fauna  u.  Flora  d.  Golfes  von  Neapel, 
xxi  [Ostracoda],  1894,  p.  8)  recommends  fixing  in  a  mixture 
of  five  parts  of  ether  and  one  of  absolute  alcohol,  followed 
by  70  per  cent,  alcohol. 

G-IESBRECHT  takes  for  marine  Copepods  a  concentrated 
solution  of  picric  acid  in  sea  water. 

KENYON  (Tufts.  Coll.  Stud.,  No.  4,  1896,  p.  80j  fixes 
Pauropoda  in  Carnoy's  acetic  alcohol  and  chloroform,  §  83, 
cuts  them  in  two  for  staining,  etc.,  and  imbeds  in  celloidin 
followed  by  tiaraffin. 

DOBOSCQ  (Arch.  Zool.  Exper.,  vi,  1899,  p.  481  ;  Journ.  Roy. 
Hie.  Soc.,  1899,  p.  544)  fixes  Chilopoda  in  a  mixture  of 
equal  parts  of  1  per  cent,  chromic  acid,  10  per  cent,  nitric 
acid,  and  95  per  cent,  alcohol,  or  in  a  mixture  of  one  part 
•of  glacial  acetic  acid  and  ten  of  absolute  alcohol. 

See  also  §  101. 

836.  Test  for  Chitin  (ZANDER,  P finger's  Arch.f.  d.  ges.  Phys., 
Ixvi,  1897,  p.  545  ;  Zeit.  f.  wiss.  Milt.,  xv,  2,  1898,  p.  214).— 
The  object  is  placed  in  water  under  a  cover- glass,  and  treated 
for  a  short  time  with  a  drop   of  freshly  prepared  solution  of 
iodine  in  iodide  of  potassium.      This  is  then  partly  removed 
with   water,    and  a  drop   of   concentrated   chloride   of    zinc 
added.      This  is  in  its  turn  removed  with  water  as  far    as 
possible,    and    the    violet    reaction    is    obtained.      Weaker 
solutions  of  zinc  chloride  may  be  taken,  but  the  reaction  is 
not  so  sharp. 

837.  Methods  for  Clearing  and  Softening  Chitin. — The  employ- 
ment of  eau  de  Javelle  or  eau  de  Labarraque,   as   suggested 


SOME    METHODS  FOE   LOWER   ANIMALS.  467 

by  Looss,  for  making  chitin  transparent  and  permeable  to 
reagents,  has  been  described,  §  556. 

LIST  (Zeit.  f.  wiss.  Mik.,  1886,  p.  212)  has  obtained  good 
results  with  Coccidae  by  treating  them  (after  hardening)  for 
eighteen  to  twenty-four  hours  with  eau  de  Javelle,  diluted 
with  four  volumes  of  water.  After  washing  out  with  water, 
they  may  be  dehydrated  with  alcohol  and  imbedded  in 
paraffin,  the  chitin  being  sufficiently  softened  to  allow  of  their 
being  penetrated  and  good  sections  being  obtained.  You 
may  stain  before  imbedding,  with  alum-carmine  or  picro- 
carmine  (five  to  six  days). 

SAZEPJN'S  method  for  antennae  of  Chilognatha  (Mem.  Acad. 
Imp.  St.  Pe'tersb.,  xxxii,  9,  1884,  pp.  11,  12)  consists  in 
steeping  antennae  (that  have  been  dehydrated  with  alcohol) 
for  twenty-four  hours  in  chloroform  containing  a  drop  of 
fuming  nitric  acid  (shake  occasionally). 

See  also  the  depigmentation.  processes,  §§  575  to  582,  and 
BETHE'S  method,  §  843. 

838.  Eyes  of  Arthropods. — For  the  methods  of  LANKESTEK 
and  BOURNE  (Quart.  Journ.  Mic  Sci.,  1883,  p.  180  :  Limulus)  ; 
HICKSON  (ibid.,  1885,  p.  243  :  Musca)  ;  PARKER  (Bull.  Mus. 
Harvard  Coll.,  xx,  1890,  p.  1;  Zeit.  f.  wise.  Milt.,  viii,  1891, 
p.  82  :  Homarus)  see  previoiis  editions. 

In  a  later  paper  (Mitth.  Zool.  Stat.  Neapel,  xii,  1895,  p.  1 ; 
Zeit.  f.  iviss.  Mik.,  xii,  4,  1896,  p.  496)  PARKER  describes  the 
application  of  the  methylen  blue  method  to  the  study  of  the 
retina  and  optic  ganglia  in  Decapods,  especially  in  Astacus. 
He  injected  0*1  c.c.  of  a  0'2  per  cent,  solution  into  the 
ventral  sinus.  After  twelve  to  fifteen  hours  the  animals 
were  killed,  the  ganglia  quickly  dissected  out,  and  the  stain 
fixed  as  described,  §  329. 

For  his  method  for  eyes  of  Scorpions  see  §  582. 

For  the  methods  of  PDRCELL  for  the  eyes  of  Phalangida 
see  Zeit.  f.  wiss.  Zool.,  Iviii,  1894,  p.  1;  Zeit.  f.  wise.  Mik., 
xii,  1,  1895,  p.  44.  He  has  the  following  stain.  The 
cephalothorax  is  removed  and  brought  for  twenty  minutes 
into  50  per  cent,  alcohol  warmed  to  45°  or  50°  C.,  and 
saturated  with  picric  acid.  The  pigment  dissolves  in  this 
solution  and  stains  the  nuclei  and  some  other  parts  of  the 
rhabdoms,  so  that  no  further  stain  is  required. 


468  CHAPTER    XXX IV. 

KOSENSTADT  (Arch,  mik.  Anat.,  xlvii,  1896,  p.  748)  fixes  eyes, 
of  Decapods  in  a  warm  mixture  of  three  parts  concentrated 
sublimate  solution  and  one  part  liquid  of  Perenyi,  and  de- 
pigmentates  them  in  a  mixture  of  three  parts  each  of  nitric 
and  hydrochloric  acid  and  100  of  water,  warmed  to  56°  C. 
for  a  few  hours. 

VIALLANES  (Ann.  Sci.  Nat.,  xiii,  1892,  p.  354;  Journ.  Roy. 
Mic.  Soc.,  1893,  p.  260)  fixes  eyes  of  Palinurus  in  5  per  cent, 
sublimate  with  5  per  cent,  acetic  acid,  washes  out  in  70  per 
cent,  alcohol,  depigmentates  in  a  mixture  of  equal  parts  of 
alcohol,  glycerin,  and  water,  through  which  chlorine  gas  is. 
led,  puts  for  twelve  hours  into  1  per  cent,  solution  of  cupric 
sulphate,  washes  for  five  to  six  hours  in  distilled  water,  and 
stains  for  twelve  hours  in  a  fresh  solution  of  one  part  haema- 
toxylin  in  100  of  absolute  alcohol  and  300  of  distilled  water. 
He  then  puts  them  back  for  the  same  time  into  the  copper 
solution,  washes,  and  passes  through  alcohol  and  makes 
paraffin  sections.  The  sections  may  be  afterwards  stained 
with  safranin. 

839.  Brain  of  Bees. — KENYON    (Journ.    Comp.    NeuroL,    vi 
1896,  p.  137;  Journ.  Roy.  Mic.  Soc.,  1897,  p.  80)  treats  them 
by  the  G-OLGI  process    (seldom  successful),   or  hardens  in  a 
mixture  of  one  part  formol  and  two  of  5  per  cent,  sulphate  of 
copper,  followed  by  staining  in  Mallory's  phospho-molybdic 
haematoxylin,  §  259. 

840.  Ventral  Cord. — BINET  (Journ.  de  I' Anat.  et  de  la  Phys., 
xxx,  1894,  p.  469)  fixes  the  ganglia  of  Hexapods  either  in 
liquid  of  Flemming,  or  in  Viallanes's  sublimate,  §  838,  treats 
them  with  his  copper  hsematoxylin,  §  838,  and  makes  paraffin 
sections,  which  he  stains  with  safranin. 

841.  Injections    (Arachnida    and    Crustacea   especially). — 
AIME    SCENEIDEE    (Toblettes  Zool.,  ii,   ]892,    p.    123)    recom- 
mends lithographic  Indian  ink,  the  animals  being  narcotised 
with    chloroform,    then     injected    and    thrown    into    strong 
alcohol. 

CAUSARD  (Bull.  Sc.  France  Belg.,xxix,  1896,  p.  16)  injects 
spiders  with  Indian  ink,  brings  them  into  alcohol,  and  thence 
for  at  least  a  day  into  water  containing  a  few  drops  of 


SOME   METHODS    FOB   LOWER   ANIMALS. 

ammonia,  which  facilitates  dissection  (VoGT  and  YUNG,  Traite 
d'Anat.  Comp.,  ii,  p.  203). 

842.  Arctiscoida  (DOYERE,  Arch.  f.  mik.  Anat.,  1865,  p.  105).— Exa- 
mination of  living  animals  after  partial  asphyxiation  in  boiled  water.  See 
previous  editions. 

843.  BETHE'S  Stain  for  Chitin  (Zool.  Jahrb.,  Abth.  f.  Anat., 
viii,  1895,  p.  544;  Zeit.  f.  wiss.  Mik.,  xii,  4,  1896,  p.  498). 
— Desirous  of  staining  the  chitinous  hairs  and  plates  of  the 
otocyst  of  Mysis,  BETHE   found  advantage   in  employing  a 
process  in  which  anilin  black  is  produced  on  the  tissue  itself. 
Anilin  black  is  a  product  of  the  oxidation  of  anilin  hydro- 
chloride.      BETHE  proceeds  as  follows  : — Series    of  sections 
mounted  on  a  slide  are  put  for  three  or  four  minutes  into  a 
freshly  prepared  10  per  cent,  solution  of  anilin  hydrochloride, 
to  which  has  been  added  one  drop  of  hydrochloric  acid  for 
every  10  c.c.      They  are  then  rinsed  in  water,  and  the  slide 
is  put  with  the  sections  downwards  into  10  per  cent,  solution 
of  bichromate  of  potash.       The  stain  quickly  begins  to  show 
itself,  but  is  at  first  in  general  not  sufficiently  intense.      The 
process  is  then  repeated  until  the  desired  intensity  of  stain 
is  obtained,  care  being  taken  to  rinse  the  sections  well  with 
water   after   each   of  the  operations,  in  order  to  avoid   the 
formation  of  precipitates.     The   stain  is  at  first  green,  but 
becomes  blue  in  tap  water  or  alcohol  containing  ammonia. 

The  same  paper  contains  a  hint  concerning  the  preparation 
of  telsons  for  section-cutting.  They  are  put  for  eight  to 
fourteen  days  into  40  per  cent,  alcohol,  to  which  nitric  acid 
is  gradually  added,  so  that  by  the  end  of  that  time  they 
have  been  brought  into  alcohol  containing  20  per  cent,  of  the 
acid.  This  softens  the  chitin,  and  somewhat  breaks  down 
the  structure  of  the  otolith,  so  that  good  sections  through  it 
are  occasionally  obtained. 

Vermes. 

844.  Enteropneusta.— Lo  BIANCO  (op.  cit.,p.  460)  fixes  with 
picro-sulphuric  acid   or  0'5   per  cent,   chromic    acid,   with 
previous  narcotisation  with  alcohol  if  desired. 

845.  Myzostoma.—  WHEELER  (Mitth.  Zool.  Stat.  Neapel,  xii, 


470  CHAPTER  XXXIV. 

1896,  p.  227)  fixes  with  sublimate  or  picro-acetic  acid,  and' 
stains  sections  with  iron  hsematoxylin  followed  by  saturated 
aqueous  solution  of  Orange  Gr. 

846.  Chsetopoda :  Fixation. — Lumlricus  may  be  anaesthetised 
by  putting  the  animals  into  water  with  a  few  drops  of  chloro- 
form. PEEEIEE  prefers  not  to  let  the  chloroform  act  directly 
in  solution  on  'the  animals,  but  to  put  them  into  water  in  a 
shallow  dish,  set  up  a  watch  glass  with  chloroform  in  the- 
corner  of  it,  and  cover  the  whole.  In  half  an  hour  the  worms 
will  be  more  or  less  narcotised,  and  if  allowed  to  remain  will 
die  in  a  state  of  extension. 

CEEFONTAINE  (Arch,  de  BioL,  x,  1890,  p.  327  ;  Zeit.f.  wise. 
Mik.,  viii,  2,  1891,  p.  210)  much  recommends  curare,  ad- 
ministered by  interstitial  injection  of  a  dose  of  about  2  c.c. 
of  a  1  :  500  solution.  The  animal  should  afterwards  be  put 
into  water,  and  after  a  quarter  of  an  hour  will  be  found 
dead. 

In  order  to  kill  Criodrilus  lacuum,  COLLIN  (Zeit.  f.  wiss. 
ZooL,  xlvi,  1888,  p.  474)  puts  the  animals  into  a  closed  vessel 
with  a  little  water,  and  hangs  up  in  it  a  strip  of  blotting- 
paper  soaked  in  chloroform.  KUKENTHAL  (Die  mik.  Technik,. 
1885  ;  Zeit.  f.  wiss.  M.ik.t  1886,  p.  61)  puts  Annelids  into  a 
glass  cylinder  filled  with  water  to  the  height  of  10  centimetres, 
and  then  pours  70  per  cent,  alcohol  to  a  depth  of  one  to  two 
centimetres  on  to  the  water.  The  animals  will  be  found  suffi- 
ciently narcotised  for  fixation  in  from  four  to  eight  hours. 
For  Opheliada3  he  also  employs  0*1  per  cent,  of  chloral  hydrate 
in  sea  water. 

Many  marine  Chastopoda  may  be  successfully  narcotised 
(S.  Lo  BIANCO)^  in  sea  water  containing  5  per  cent,  of  alcohol, 
or  by  means  of  the  mixture  §  16. 

The  Polychseta  sedentaria  offer  the  difficulty  of  a  complex 
and  very  contractile  branchial  apparatus.  They  may  some- 
times be  satisfactorily  fixed  by  bringing  them  rapidly  into 
corrosive  sublimate.  Cold,  not  hot  solutions  should  be 
taken,  as  heat  frequently  shrivels  up  the  branchiae.  The 
species  of  Polychssta  errant ia  that  offer  a  contractile  branchial 
apparatus,  as  Eunice  and  Onuphis,  may  be  treated  in  the 
same  way. 

S.  Lo  BIANCO  advises  killing  Chaetopterjdae,  Sternaspidas,. 


SOME  METHODS  FOE  LOWER   ANIMALS.  4?1 

Spirographis,  Protula,  by  putting  them  for  half  an  hour  into 
1  per  cent,  chromic  acid.  I  have  satisfied  myself  that  good 
show  specimens  can  be  obtained  in  this  way;  but  I  doubt 
the  histological  preservation  of  the  parts  being  so  good  as 
with  sublimate  specimens.  Some  of  the  sedentaria  may  be 
got  protruded  from  their  tubes  by  leaving  them  for  some 
hours  in  0'  1  per  cent,  chloral  hydrate  in  sea  water  (S.  Lo 
BIANCO). 

See  also  §  12  (lemon  juice),  and  the  methods  §§  18  to  24 
43,  and  53. 

RIEVEL  (Zeit.  wiss.  Zool.,  Ixii,  1896,  p.  292)  fixes  Ophryo- 
trocha  in  extension  in  hot  liquid  of  Lang  (§67),  five  to  eight 
minutes,  and  Lumbricus  in  hot  alcoholic  sublimate  or  hot 
picro- sulphuric  acid,  ten  to  fifteen  minutes. 

For  Elsie's  methods  for  Capitellidae  see  Fauna  u.  Flora  Golf. 
Neapel,  xvi,  1887,  p.  295,  or  Grtindziige,  p.  394. 

847.  Staining. — For  the  staining  of  small  Annelids  entire  I 
find  carmalum  gives  very  good  results,  I  think  better  than 
borax-carmine  or  paracarmine. 

848.  Blood-vessels  of  Annelids   (KUKENTHAL,   Zeit.  f.   wiss. 
Mik.,  1886,  p.  dl). — The  animals  should  be  laid  open  and  put 
for  two  or  three  hours  into  aqua  regia  (4  parts  of  nitric  acid 
to  2  of  hydrochloric  acid).      The  ramifications  of  the  vessels 
will  then  be  found  to  be  stained  black,  the  rest  of  the  pre- 
paration yellow. 

849.  Nerves  of  Annelids.— The  methylen  blue  method  and 
the  bichromate  of  silver  method  of  Golgi  (the  rapid  method). 
For  the  latter  see  v.  LENHOSSEK  (Arch.  f.  mik.  Anat.,  xxxix, 
p.  102;  Zeit.f.  wi**.  A/tfc.,  ix,  3,  1893,  p.  432). 

See  also  M.  LEWIS,  Anat.  Anz.,  xii,  1896,  p.  292 ;  and  the 
methods  of  APATHY,  §§  358,  707. 

850.  Cleansing  Intestine  of  Lumlricns  (KUKENTHAL,  Journ. 
Roy.  Mic.  8oc.,  1888,  p.  1044).— Put  the  animals  into  a  tall 
glass  vessel  which  has  been  filled  up  with  bits  of  moistened 
blotting-paper.     They  gradually  evacuate  the  earthy  particles 
from  the  gut,  and  fill  it  instead  with  paper. 


472  CHAPTER    XXXIV. 

VOGT  and  YUNG  (Traite  d'Anat.  Comp.  Prat.,  v)  recommend 
coffee-grounds  instead  of  paper;  paper  becomes  rather  hard 
when  imbedded,  whereas  coffee-grounds  cut  fairly  well. 

JOEST  (Arch.  Entwicklungsmech.,  v,  1897,  p.  425)  simply 
keeps  the  worms  for  a  few  days  in  moist  linen,  and  finds  the 
gut  empty. 

851.  Hirudinea. — For  the  methods  of  killing  see  those  given 
for  Lumbricus  in  §  846,  also  §§  18  to  24,  and  53. 

WHITMAN  (Meth.  in  mic.  Anat.,  p.  27)  recommends  that  they 
be  killed  with  sublimate. 

I  have  obtained  better  results  myself  by  narcotising  with 
carbonic  acid  (§  24),  and  fixing  with  liquid  of  Flemming.  I 
have  also  found  that  lemon  juice  kills  them  in  a  state  of  very 
fair  extension.  Carmalum  I  find  excellent  for  staining  entire 
animals.  Ehrlich-Biondi  mixture  sometimes  gives  fine  results 
with  sections. 

GRAF  (Jen.  Zeit.,  1893,  p.  165)  states  that  he  has  obtained 
good  results  by  narcotising  with  decoction  of  tobacco. 

Injection. — WHITMAN  (Amer.  Natural.,  1886,  p.  318)  states 
that  very  perfect  natural  injections  may  often  be  obtained 
from  leeches  that  have  been  hardened  in  weak  chromic  acid 
or  other  chromic  liquid.  He  considers  that  these  injections 
are  the  best  for  the  purpose  of  the  study  of  the  circulatory 
system  by  means  of  sections. 

JACQUET  (Mitth.  Zool.  Stat.  Neapel,  1885,  p.  298)  advises 
that  leeches  be  put  into  water  with  a  very  small  quantity  of 
chloroform;  they  soon  fall  to  the  bottom  of  the  vessel  and 
remain  motionless.  They  should  be  allowed  to  remain  a  day 
or  two  in  the  water  before  injecting  them. 

852.  Gephyrea.— VOGT  and  YUNG  (Anat.  Comp.  Prat.,  p.  373) 
direct  that  Siphunculus  midus  be  kept  for  some  days  in  per- 
fectly clean  basins  of  sea  water,  in  order  that  the  intestine 
of  the  animals  may  be  got  free  from  sand,  which  would  be  an 
obstacle  to  section-cutting,  and  then  anaesthetised  with  chloro- 
form, under  which  treatment  they  die  extended,  and  may  be 
fixed  as  desired. 

WAKD  (Bull.  Mus.  Comp.  Zool.,  Cambridge,  Harvard  Coll., 
xxi,  3,  p.  144)  found  the  best  plan  was  to  put  the  animals  into 
a  shallow  dish  with  sea  water  and  pour  5  per  cent,  alcohol  in 


SOME    METHODS    FOR    LOWER  ANIMALS.  473 

a  thin  film  on  to  the  surface  of  the  water.  After  four  to  eight 
hours,  if  the  animals  make  no  contractions  on  being  stimu- 
lated, they  may  be  removed  to  50  per  cent,  alcohol. 

S.  Lo  BIANCO  says  killing  with  0*5  per  cent,  chromic  acid 
or  with  0*1  per  cent,  chloral  hydrate  in  sea  water  may  be 
tried,  but  either  method  is  uncertain.  Phaxcolosoma  and 
Phoronis  should  be  treated  by  the  alcohol  method. 

APEL  (Zeit.  f.  wiss.  ZooL,  xlii,  1885,  p.  461)  says  that 
Priapulus  and  Holier yptus  can  only  be  satisfactorily  killed  by 
heat.  The  animals  may  either  be  put  into  a  vessel  with  sea 
water  and  be  heated  on  a  water-bath  to  40°  C.;  or  they  may 
be  thrown  as  rapidly  as  possible  into  boiling  water,  which 
paralyses  them  so  that  they  can  be  quickly  cut  open  and 
thrown  into  -J-  per  cent,  chromic  acid  or  picro-sulphuric  acid. 

853.  Rotatoria. — By  far  the  most  important  method  for  the 
study  of  this  group  consists  in  the  observation  of  the  living 
animals.  For  quieting  them  WEBER  (Arch,  de  BioL,  viii,  4, 
1888,  p.  713)  finds  that  of  all  the  reagents  he  tried,  2  per 
cent,  solution  of  hydrochlorate  of  cocaine  gave  the  best 
results.  Warm  water  gave  him  good  results  for  large 
species,  such  as  those  of  Hydatina  and  Brachionus. 

HARDY  (Journ.  Roy.  Mic.  Soc.,  1889,  p.  475)  recommends 
thick  syrup  added  drop  by  drop  to  the  water.  HUDSON  (ibid., 
p.  476)  mentions  weak  solution  of  salicylic  acid. 

HOFER'S  hydroxylamin  method  has  been  given,  §  20,  and 
TULLBERG'S  chloride  of  magnesium  method,  §  21  ;  the  pro- 
cesses of  EISMOND  and  JENSEN,  §  879,  maybe  tried.  Methy- 
len  blue,  §  323,  may  be  found  useful. 

Permanent  preparations  may  be  made  by  the  method  of 
RODSSELET  (Journ.  Quekett  Mic.  Club,  v,  March,  1895,  p. 
The   animals   are  got   together   in    a    watc-h  glass    and   are 
narcotised  by  adding  to  the  water  at  intervals  a  few  drops 
of  the  following  mixture  : 

Hydrochlorate  of  cocaine  2  per  cent,  solution  .  3  parts. 
Methylated  spirit    .  •    l  Part< 

Water        .  •   6  Part*' 

They  are  watched  under  a  dissecting  micro* 
the  moment  when  the  cilia  have  ceased  to  beat,  or  »i 
to   be  on  the  point  of  ceasing   to  beat,  they  are 
adding  a  drop  of  liquid  of  Flemming  or  of  i  per  cent   osmic 


474  CHAPTER    XXXTV. 

acid.  The  fixing  agent  is  allowed  to  act  for  half  a  minute 
or  less,  after  which  the  animals  are  taken  out  with  a  pipette, 
and  thoroughly  washed  by  passing  them  through  two  or 
three  watch  glasses  of  distilled  water.  They  are  then 
definitely  mounted  in  2^  per  cent,  solution  of  formaldehyde 
(formol  2^  parts,  distilled  water  37^  parts).  See  also  §  455. 

For  some  details  concerning  variations  of  this  method 
adapted  to  the  preservation  of  the  different  forms,  see  the 
paper  quoted. 

ZOGEAF  (Gomptes  Rend.,  cxxiv,  1897,  p.  245;  Zeit.f.wiss. 
Milt.,  xiv,  1897,  p.  380  ;  Journ.  Roy.  Mic.  Soc.,  1897,  p.  173) 
narcotises  as  ROUSSELET,  but  without  the  spirit,  fixes  with 
osmic  acid  for  two  to  four  minutes,  then  replaces  this  by  raw 
pyroligneous  acid  diluted  with  eight  to  ten  volumes  of  water, 
and  after  five  to  ten  minutes  washes  in  several  changes  of 
water,  and  passes  through  successive  alcohols  into  glycerin 
or  balsam. 

See  also  §25  (VoLK). 

CONSEE  (Trans.  Amer.  Mic.  Soc.,  xvii,  1896,  p.  310)  narcotises  with 
cocaine,  and  fixes  with  20  per  cent,  formol  followed  by  0*5  per  cent,  chromic 
acid. 

854.  Acanthocephali. — It  is  very  difficult  to  kill  Echino- 
rhynci  so  as  to  have  the  animals  duly  extended  and  the 
tissues  well  preserved.  Neither  corrosive  sublimate  nor 
strong  osmic  acid  will,  as  a  rule,  attain  this  end,  even  after 
preliminary  intoxication  with  tobacco  smoke  or  chloroform, 
the  animal  thus  treated  dying  contracted. 

HAMANN,  however  (Jen.  Zeit.  f.  Naturw.,  xxv,  1890,  p. 
113;  Zeit.  f.  wiss.  Mik.,  viii,  2,  1891,  p.  209),  has  succeeded 
with  sublimate;  and  also  with  alcohol  containing  a  little 
platinum  chloride. 

SAEOTIGEN  (Morphol.  Jahrb.,  x,  1884,  120;  Journ.  Roy. 
Mic.  Soc.  [N.  S.],  v,  1885,  p.  147)  obtained  the  best  results 
by  killing  gradually  with  O'l  per  cent,  osmic  acid;  the 
animals  placed  in  this  contract  during  the  first  hours,  but 
stretch  out  again  and  die  fully  extended. 

Another  method  of  killing  is  treatment  with  O'l  per  cent 
chromic  acid  ;  Echinorhynci  live  for  days  in  it,  but  eventu- 
ally die  fully  extended. 

KAISKR   (Biblioth.   Zool.,   H.  vii,    1   Halfte,   1891  ;   Zeit.  /.. 


SOME   METHODS    FOR    LOWER    ANIMALS.  475 

wi*8.  Mik.,  viii,  3,  1891,  p.  363)  found  that  a  saturated 
aqueous  solution  of  cyanide  of  mercury,  warmed  to  45°  to 
50°  C.,  and  allowed  to  act  for  from  fifteen  m  sixty  minutes, 
and  then  washed  out  with  70  per  cent,  alcohol,  wsis  the  best 
of  all  fixing  media  for  Acanthocephali. 

He  also  found  the  following  mixture  excellent  : 

Picric  acid     v,          ...  1  gramme. 

Cone,  sulphuric  acid            .           .  10  gramim-. 

Chromic  acid   ....  1  gramme. 

Water    .                                         .  1000  grammes. 

To  be  warmed  to  55°  C.,  allowed  to  act  for  fifteen  to 
twenty  minutes,  washed  out  for  five  to  ten  minutes  with 
hot  water,  and  afterwards  for  some  days  in  60  per  cent, 
alcohol. 

855.  Nematodes. — The  extremely  impermeable  cuticle  of 
these  animals  is  a  great  obstacle  to  preparation.  According 
to  Looss  (Zool.  Anz.,  1885,  p.  318)  this  difficulty  may  be 
overcome  in  the  manner  described  in  §  556. 

For  fixing,  most  recent  authors  recommend  sublimate 
solutions;  chromic  solutions  seem  to  have  a  tendency  to 
make  the  worms  brittle. 

But,  according  to  ZUE  STEASSEN  (Zeit.  f.  u-iss.  Zool.,  liv> 
p.  655),  Bradynema  rigidum  ought  to  be  fixed  for  at  least 
twelve  hours  in  mixture  of  Flemming. 

AUGSTEIN  (Arch.  f.  Naturg.  Jahrg.,  Ix,  1,  1894,  p.  255  ; 
Zeit.  /.  wiss.  Mik.,  xii,  2,  1895,  p.  227)  found  that  for 
Strongylus  filaria  the  best  fixing  agent  was  Mayer's  picro- 
nitric  acid. 

YEJDOVSKY  (Zeit.  wiss.  Zool.,  Ivii,  1894,  p.  645)  advi>«-< 
for  Gordius  0'5  per  cent,  chromic  acid  (twenty-four  hours). 

Lo  BIANCO  (loc.  cit.,  p.  462)  employs  for  marine  forma 
concentrated  sublimate  or  picro-sulphuric  acid. 

COBB  (ante,  p.  4)  uses  his  differentiator  for  bringing 
through  the  various  media  after  fixation. 

Staining  is  frequently  difficult,  and  sometimes  alcoholic 
carmine,  §  229,  is  the  only  thing  that  will  give  fair  result.-,. 

BBAUN  (see  Journ.  Roy.  Mic.  Soc.,  1885,  p.  897)  recommend*  that  small 
unstained  Nematodes  be  mounted  in  a  mixture  of  2t>  parts  gelatin,  100 
parts  glycerin,  120  parts  water,  and  2  parts  carbolic  acid,  which  is  melted  at 


476  CHAPTER    XXXIV. 

the  moment  of  using.     Canada  balsam,  curiously  enough,  is  said  to  some- 
times make  Nematodes  opaque. 

Demonstration  of  living  Trichinse  (Barnes,  Amer.  Mon.  Mic.  Journ., 
xiv,  1893,  p.  104;  Journ.  Roy.  Mic.  Soc.,  1893,  p.  406).— A  piece  of 
trichinised  muscle  of  the  size  of  a  pea  should  be  placed  in  a  bottle  in  a 
mixture  of  3  gr.  of  pepsin,  2  dr.  of  water,  and  2  minims  of  hydrochloric 
acid.  The  whole  should  be  kept  at  body  temperature  for  about  three  hours 
with  occasional  shaking.  The  flesh  and  cysts  being  dissolved,  the  fluid  is 
poured  into  a  conical  glass,  and  allowed  to  settle ;  the  trichinae  are  drawn 
off  from  the  bottom  with  a  pipette,  got  on  to  a  slide  with  water,  and  exa- 
mined on  a  hot  stage. 

GEAHAM  (Arch.  mik.  Anat.,  1,  1897,  p.  216)  isolates  Trichinae  by  mace- 
rating for  one  or  two  days  in  2  per  cent,  acetic  acid,  staining  with  aceto- 
carmine,  and  teasing. 

856.  Nemertina. — After  considerable  experience  of  this 
difficult  group  I  have  to  say  that  I  know  of  no  method  of 
fixation  that  will  certainly  give  good  results.  My  best 
results  have  always  been  obtained  with  cold  saturated 
sublimate  solution,  acidified  with  acetic  acid.  I  have  tried 
most  of  the  other  usual  fixing  agents,  such  as  the  osmic  and 
chromic  mixtures,  and  do  not  recommend  them  for  this 
group,  for  they  seem  (the  chromic  mixtures  and  perchloride 
of  iron  in  particular)  to  act  as  irritants,  and  provoke  such 
violent  muscular  contractions  that  the  whole  of  the  tissues 
are  crushed  out  of  shape  by  them.  And,  besides,  they  do 
not  kill  as  quickly  as  sublimate. 

I  have  found  it  a  good  plan  to  decapitate  the  animals  (in 
the  larger  forms),  cut  them  up  quickly  into  lengths  (not  too 
long),  and  throw  these  sharply  into  the  sublimate,  the 
muscular  contractions  being  less  energetic  in  segments  that 
are  no  longer  in  connection  with  the  cerebral  ganglia. 

Perhaps  a  better  method  than  this  will  be  found  in  the 
simple  process,  suggested  to  me  by  Prof.  DU  PLESSIS,  of  fixing 
with  hot  (almost  boiling)  water.  On  the  few  occasions  on 
which  I  have  tried  it  the  animals  have  died  in  extension, 
without  vomiting  their  proboscis ;  and  I  think  it  is  certainly 
worth  trial,  especially  for  the  larger  forms. 

I  have  tried  FOETTINGER'S  chloral  hydrate  method  (§  18). 
My  specimens  died  fairly  extended,  but  vomited  their  pro- 
boscides.  According  to  S.  Lo  BIANCO  (loc.  cit.,  p.  461) 
narcotisation  with  a  solution  of  0*1  to  0*2  per  cent,  in  sea 
water  for  six  to  twelve  hours  is  useful. 


SOME    METHODS   FOR   LOWER  ANIMALS.  477 

DENDY  (see  Journ.  Roy.  Mic.  £oc.,  189:3,  p.  116)  has 
succeeded  with  Geonemertes  by  exposing  it  for  half  a  mi  nut  r 
to  the  vapour  of  chloroform. 

For  staining  fixed  specimens  in  toto  I  hold  that  it  is  well- 
nigh  necessary  to  employ  alcoholic  stains,  for  even  the  most 
delicate  species  are  not  satisfactorily  penetrated  by  watery 
stains  in  any  reasonable  lapse  of  time.  Borax-carmine  or 
Mayer's  alcoholic  carmine  may  be  recommended ;  not  so 
cochineal  or  ha3matoxylin  stains,  on  account  of  the  energy 
with  which  they  are  held  by  the  mucin  which  in  general 
exists  in  such  great  abundance  in  the  skin  of  these  animals. 

Sections  by  the  paraffin  method,  after  penetration  with 
oil  of  cedar  (chloroform  will  fail  to  penetrate  sometimes 
after  the  lapse  of  weeks). 

BURGER  (Fauna  n.  Flora  Golf.  Neapel,  xxii,  1895,  p.  443) 
studies  the  nervous  system,  nephridia,  skin,  muscle,  and 
intestine  by  the  intra  vitam  methylen-blue  method.  He 
injects  the  animals  with  0*5  per  cent,  solution  in  distilled 
water,  or  0*5  per  cent,  salt  water,  and  allows  them  to  lie  for 
six  to  twelve  hours  or  more  in  moist  blotting-paper. 

He  also  employs  maceration  in  one  third  alcohol,  or  the 
Hertwigs'  medium,  §  538. 

For  his  other  methods  see  the  paper  quoted,  or  Grundziige, 
p.  399. 

See  also  MONTGOMERY  (Zool.  Jahrb.,  Abth.  Morph.,  x,  1897,  p.  6 ; 
Grundzuge,  p.  399). 

857.  Cestodes. — This  group  must  of  course  be  chiefly  studied 
by  the  usual  section  methods.  As  pointed  out  by  VOGT 
and  YUNG  (Trait*  d'Anat.  Comp.  Prat.,  p.  204),  the  observa- 
tion of  the  living  animal  may  be  of  service,  especially  in  the 
study  of  the  excretory  system.  And,  as  shown  by  PINTNER, 
Tsenias  may  be  preserved  alive  for  several  days  in  common 
water  to  which  a  little  white  of  egg  has  been  added. 

LONNBERG  (Centralb.f.  BaUerioL  u.  Parasitenk.,  xi,  1892, 
p.  89 ;  Journ.  Roy.  Mic.  Soc.,  1892,  p.  281)  has  kept  Triseno- 
phorus  nodulosus,  a  parasite  of  the  pike,  alive  for  a  month  in 
a  slightly  acid  pepsin-peptone  solution  containing  from  3  to 
4  per  cent,  of  nutritive  matter,  and  less  than  1  per  cent,  of 

NaCl. 

DE  FILIPPI  (Att.  Accad.  Lined,  vii,  1894,  p.  250 ;  Zeit.  f. 


478  CHAPTER   XXXIV. 

wiss.  Mik.,  xiii,  1897,  p.  484)  finds  that  for  Tdenia  bothrio- 
plitis  the  best  fixative  is  saturated  aqueous  picric  acid  for 
about  seven  hours ;  sublimate  gave  bad  results. 

TOWER  (Zool.  Anz.,  xix,  1896,  p.  323;  Journ.  Roy.  Hie. 
Soc.,  1896,  p.  571)  fixes  Cestodes  in  a  picro-platin-osmic 
mixture  (stronger  than  that  of  0.  vom  Rath,  §  97)  for 
ten  hours,  then  treats  for  several  hours  with  crude  pyro- 
ligneous  acid,  and  lastly  with  alcohol,  and  imbeds  in 
paraffin. 

ZERNECKE  (Zool.  Jahrb.,  Abth.  f.  Anat.,  ix,  1895,  p.  92 ;  Zeit.  f.  wiss. 
Mik.,  xii,  4,  1896,  p.  494)  has  employed  with  success  the  bichromate  of 
silver  impregnation  of  GOLGI.  He  kills  Ligula  in  the  osmio-bichromic 
mixture  (4 : 1),  impregnates  as  usual,  makes  sections  in  liver,  and  treats 
them  by  the  hydroquinon  process  of  KALLIUS.  Besides  the  peripheral  and 
central  nervous  system,  muscle-fibres,  parenchyma  cells,  and  the  excretory 
vascular  system  are  impregnated. 

He  has  also  obtained  good  results  by  the  methylen-blue  method. 

BLOCHMANN  (Biol.  Centralb.,  xv,  1895,  p.  14;  Zeit.  f.  wiss.  Mik.,  xii, 
1895,  p.  226)  recommends  for  the  nervous  system  the  bichromate  and 
sublimate  method  of  GOLGI. 

See  also  KOHLEE,  Zeit.  wiss.  Zool.,  Ivii,  1894,  p.  386  (stretches  Taeniae 
round  a  glass  plate  or  on  cork,  and  fixes  with  5  per  cent,  sublimate). 

858.  Trematodes  (FISCHER,  Zeit.  f.  wiss.  Zool.,  1884,  p.  1). — 
Opisthotrema  cochleare  may  be  mounted  entire  in  balsam. 
For  sectioning,  FISCHER  recommends  imbedding  in  a  mass 
made  by  dissolving  15  parts  of  soap  in  17 '5  parts  of  96  per 
cent,  alcohol.  This  mass  melts  at  about  60°  C.,  penetrates 
very  rapidly,  and  solidifies  very  quickly.  The  sections  should 
be  studied  in  glycerin. 

WRIGHT  and  MACALLUM  (Journ.  of  Morph.,  i,  1887,  p.  1) 
find  that  Sphyranura  is  for  most  purposes  best  fixed  in 
liquid  of  Flemming,  and  stained  with  alum-cochineal. 

Lo  BIANCO  (l-oc.  cit.,  p.  460)  fixes  Trematodes  with  hot 
saturated  sublimate. 

Looss  (Arch.  mik.  Anat.,  1895,  p.  7)  takes  for  Bilharzia 
warm  (50°  to  60°  C.)  1  per  €ent.  sublimate  in  70  per  cent, 
alcohol. 

BETTENDOEF  (Zool.  Jahrb.,  Abth.  Morph.,  x,  1897,  p.  308)  has  had  good 
results  with  the  rapid  Golgi  method  only  on  Distoma  hepaticum,  and 
prefers  methylen  blue. 

Cercarise. — SCHWARTZ  (Zeit.  f.  iviss.  ZooL,  xliii,  1886,  p. 
•45)  found  that  the  only  fixing  agent  that  would  preserve  the 


SOME    METHODS    FOR    LOWER    ANIMALS.  479 

histological  detail  of  these  forms  was  cold  saturated  sublimate 
solution  warmed  to  35° — 40°  C. 

859.  Turbellaria. — For  Bhabdocoela,  BRADN  (Zeit.  f.  wiss. 
J//A-.,  iii,  1886,  p.  398)  proceeds  as  follows : — For  preparing 
entire  animals,  the  specimens  are  got  on  to  a  slide,  lightly 
flattened  out  with  a  cover,  and  killed  by  running  under  the 
cover  a  mixture  of  three  parts  of  liquid  of  Lang  with  one  of 
1  per  cent,  osmic  acid  solution.  Other  fixing  media  than 
that  described  were  not  satisfactory.  (BoHMio  [ibid.'],  com- 
menting on  this,  says  that  for  some  of  the  tissues,  such  as 
muscle  and  body  parenchyma,  nitric  acid  and  picro-sulphuric 
acid  are  very  useful.)  Sections  may  be  made  by  the  paraffin 
method. 

DELAGE  (Arch,  de  Zool.  exp.  et  gen.,  iv,  2,  1886 ;  Zeit.  f.  wiss.  Mik.,  iii, 
2,  1886,  p.  239  ;  Journ.  Roy.  Mic.  Soc.,  1886,  p.  1073)  strongly  recommends 
fixation  (of  Rhabdocoela  Acoela)  by  an  osmium-carmine  mixture,  for  which 
see  loc.  cit.,  or  by  concentrated  solution  of  sulphate  of  iron.  Liquid  of 
Lang  was  not  successful. 

For  staining,  he  recommends  either  the  osmium-carmine  stain  or  impreg- 
nation with  gold  (-|  formic  acid,  two  minutes  ;  1  per  cent,  gold  chloride,  ten 
minutes;  2  per  cent,  formic  acid,  two  or  three  days  in  the  dark.  It  is  well 
to  allow  an  excessive  reduction  to  take  place,  and  then  lighten  the  stain  by 
means  of  1  per  cent,  solution  of  cyanide  of  potassium). 

BOHMIG  (Zeit.  f.  wiss.  Mik.,  iii,  1886,  p.  239)  says  that  he  has  obtained 
very  instructive  images  with  Plagiostomidae  fixed  with  sublimate  and 
stained  with  the  osmium-carmine. 

GRAFF  (Organisation  d.  Turbellaria  Acoela,  Leipzig,  1891  ; 
Zeit.  f.  wits.  Mik.,  ix,  1,  1892,  p.  76)  says  that  chromo- 
aceto-osmic  acid,  followed  by  haematoxylin,  is  good  for  the 
skin,  but  will  not  afford  a  satisfactory  preservation  of  the 
Rhabdites,  which  in  Accela  and  Alloioccela  seem  to  be 
destroyed  by  swelling.  The  same  method  is  also  good  for 
the  parenchyma  of  Amphichcerus  cinereus,  Convoluta  para- 
doxa,  and  C.  sordida.  Sublimate  is  good  for  Convoluta 
Poscoffensis.  For  some  forms  it  is  important  to  avoid  picro- 
carmine,  which  destroys  the  central  parenchyma.  The 
nervous  system  may  be  investigated  by  the  methods  of 
DELAGE. 

For  Dendrocosla  sublimate  solutions,  sometimes  hot,  appear 
indicated  for  fixing.  CHICHKOFF  (Arch,  de  BioL,  xii,  1892, 
p.  438 ;  Journ.  Roy.  Mic.  Soc.,  1893,  p.  262)  recommends 


480  CHAPTEE    XXXIV. 

the  following  for  fresh- water  Dendroccela : — 2  per  cent, 
sublimate  solution  6  parts ;  15  per  cent,  acetic  acid,  4  parts  ; 
pure  nitric  acid,  2  parts  ;  14  per  cent,  chloride  of  sodium,  8 
parts  ;  and  2  per  cent,  alum,  1  part.  Note  also  the  mix- 
tures of  Lang,  §  67.  Mayer's  tincture  of  cochineal,  §  230, 
may  be  found  useful  for  the  study  of  glands,  for  which 
purpose  the  Ehrlich-Biondi  stain  may  also  be  employed. 

Lo  BIANCO  (loc.  cit.,  p.  461)  kills  Rhabdoccela  and  Dendro- 
ccela with  hot  sublimate,  throws  them  at  once  into  cold 
water,  and  then  brings  them  into  alcohol.  For  some  Poly- 
clads  the  sublimate  must  not  be  more  than  slightly  warm. 

VOIGT  (Verh.  Nat.  Ver.  Bonn,  1896,  p.  118)  kills  Planaria 
by  pouring  off  the  water  it  is  in  and  inundating  it  with  a 
mixture  of  one  part  concentrated  nitric  acid  and  three  parts 
water,  and  after  one  minute  brings  into  alcohol  of  70  to  90 
per  cent. 

KLINCKOWSTROEM  (Arch.  mik.  Anat.,  xlviii,  1897,  p.  589) 
fixes  Prosthecer&us  in  70  per  cent,  alcohol  with  4  per  cent, 
of  acetic  acid. 

JAENICHEN  (Zeit.  wiss.  Zool.,  Ixii,  1896,  p.  256)  advises 
for  Planaria,  eyes  especially,  pier o- sulphuric  acid  for  an 
hour  or  two ;  osmic  acid  is  not  good,  and  liquid  of  Miiller 
macerates.  He  stains  with  borax -carmine,  makes  sections, 
and  puts  them  for  ten  minutes  into  osmic  acid,  then  for  five 
minutes  into  pyroligneous  acid,  on  the  top  of  the  stove. 
He  macerates  the  visual  rods  in  a  mixture  of  one  part 
common  salt,  one  of  acetic  acid,  and  100  of  water.  He 
bleaches  the  pigment  of  the  eyes  with  peroxide  of  hydrogen. 


Echinodermata. 

860.  Holothurioidea. — These  animals  are  difficult  to  fix  on 
account  of  their  contracting  with  such  violence  under  the 
influence  of  irritating  reagents  as  to  expel  their  viscera 
through  the  oral  or  cloacal  aperture. 

S.  Lo  BIANCO  (loc.  cit.,  p.  459)  puts  them  into  pure  sea 
water  until  they  have  expanded  their  tentacles,  then  seizes 
them  with  forceps  or  the  fingers  behind  the  tentacles,  so  as 
to  mechanically  render  impossible  their  withdrawal,  and 
immerses  the  anterior  part  of  the  body  in  acetic  acid,  whilst 


SOME    METHODS    FOR  LOWER  ANIMALS.  481 

at  the  same  time  an  assistant  injects  90  per  cent,  alcohol 
through  the  anus. 

YOGT  and  YUNG  (Anat.  Comp.  Prat.,  p.  641)  say  that 
Cucumaria  Planci  (C.  doliolum,  Marenzeller)  is  free  from 
the  vice  of  expelling  its  intestines  under  irritation ;  but  they 
recommend  that  it  be  killed  with  fresh  water,  or  by  slow 
intoxication  with  alcohol,  chromic  acid,  or  sublimate  added 
to  the  sea  water  in  which  it  is  contained. 

Synapta  may  be  allowed  to  die  in  a  mixture  of  equal 
parts  of  sea  water  and  ether  or  chloroform  (S.  Lo  BIANCO). 

Holothurids,  Dr.  WE  BEE  informs  me,  are  admirably  pre- 
served in  formaldehyde ;  a  weak  solution  is  sufficient. 

GEEAULD  (Bull.  Mus.  Harvard  Coll.,  xxix,  1896,  p.  125 ; 
Journ.  Roy.  Hie.  Soc.,  1896,  p.  476)  paralyses  Caudina  with 
sulphate  of  magnesia,  §  21,  and  fixes  with  liquid  of  Perenyi 
(or  sublimate  for  the  ovaries).  He  demonstrates  cell-limits 
by  rinsing  epithelia  with  distilled  water  and  impregnating 
with  1  per  cent,  silver  nitrate. 

HEROCAED  (Arch.  Zool.  Expe'r.,  vii,  1899,  p.  537)  kills 
Cucumaria  by  plunging  into  a  1  per  cent,  solution  of  chloral 
hydrate  warmed  to  40°  C.,  the  anus  being  closed  by  means 
of  forceps. 

For  the  staining  of  muscles  with  methylen  blue  see  IWANZOFF,  Arch.f. 
mil:.  Anat.,  xlix,  1897,  p.  103. 

861.  Asteroidea. — HAMANN  (Beitr.  z.  Hist.  d.  Echinodermen, 
ii,  1885,  p.  2)  finds  it  best  to  inject  the  living  animal  with  a 
fixing  liquid.  The  cannula  should  be  introduced  under  the 
integument  at  the  extremity  of  a  ray,  and  the  liquid  injected 
into  the  body-cavity.  The  ambulacral  feet  and  the  branchiae 
are  soon  distended  by  the  fluid,  and  as  soon  as  it  seems  to 
have  penetrated  sufficiently  the  animal  is  thrown  into  a 
quantity  of  the  same  reagent. 

In  order  to  study  the  eyes,  with  the  pigment  preserved  in 
situ,  they  should  be  removed  by  dissection,  should  be 
hardened  in  a  mixture  of  equal  parts  of  1  per  cent,  osmic 
acid  and  1  per  cent,  acetic  acid,  and  sectioned  in  a  glycerin 
gum  mass,  or  some  other  mass  that  does  not  necessitate 
treatment  with  alcohol  (which  dissolves  out  the  pigment, 
leaving  the  pigmented  cells  perfectly  hyaline).  For  mace- 

81 


482  CHAPTER   XXXIV. 

ration  use  one  third  alcohol,  the  aceto-osmic  mixture  failing 
to  preserve  the  rods  of  the  pigmented  cells. 

Formaldehyde  is  not  to  be  recommended  for  the  preser- 
vation of  Asteroidea  (WEBER)  . 

See  also  Lo  BIANCO,  loc.  cit.,  p.  458  (he  kills  Brisinga 
with  absolute  alcohol)  ;  also  §§  15,  18. 

862.  Ophiuridea    should    in    general    be   killed    in    fresh 
water  if  it  be   desired  to   avoid  rupture   of  the   rays    (DE 
CASTELLARNAU,  La  Est.  Zool.  de  Napoles,  p.  135). 

Lo  BIANCO  (loc.  cit.,  p.  458)  kills  small  forms  with  weak 
alcohol,  Ophiopsila  with  absolute  alcohol,  and  Ophiomyxa 
with  0*5  per  cent,  chromic  acid. 

Russo  (Ricerche  Lab.  Anat.  Roma,  iv,  1895,  p.  157)  fixes 
Ophiothrix  for  an  hour  or  two  in  0*5  per  cent,  osmic  acid 
and  then  decalcifies  in  solution  of  Miiller  for  six  to  ten  days. 
Or  he  fixes  for  three  minutes  in  a  mixture  of  two  parts 
concentrated  sublimate  solution,  one  part  70  per  cent, 
alcohol,  and  one  part  acetic  acid  (sp.  gr.  l'06j,  and  decal- 
cifies in  Miiller  or  in  70  per  cent,  alcohol  with  10  per  cent, 
of  acetic  acid.  He  stains  with  paracarmine. 

863.  Echinoidea. — I  advise  that  they  be  killed  by  injection 
of  some  fixing  liquid.      For  preservation,  formaldehyde  has 
proved    admirable   in   all  respects,   and   greatly  superior  to 
alcohol  (WEBER). 

Lo  BIANCO  (loc.  cit.,  p.  458)  kills  by  pouring  over  them 
(mouth  upwards)  a  mixture  of  ten  parts  acetic  acid  and  one 
of  1  per  cent,  chromic  acid,  and  brings  at  once  into  weak 
alcohol.  Or  he  makes  two  holes  in  the  shell,  lets  the  water 
run  out  and  alcohol  run  in. 

Sections  of  spines  may  be  made  by  grinding,  see  §  176. 

864.  Crinoidea. — Lo  BIANCO  (loc.  cit.,  p.  458)  fixes  Antedon 
rosacea  with    70   per   cent,  alcohol,  A.  phalangium  with  90 
per  cent. 

865.  Larvae  of  Echinodermata    (from    instructions    written 
down  for  me  by  Dr.  BARROIS). — In  order  to  a  fruitful  study 
of  the  metamorphoses  of  the  Echinoidea  and  Ophiuridea  it 
is  necessary  to  obtain  preparations  that  give  distinct  images 


SOME    METHODS   FOR    LOWER    ANIMALS.  483 

of  the  different  organs,  and  show  the  calcareous  skeleton 
preserved  intact  (a  point  of  considerable  importance,  since 
this  skeleton  frequently  affords  landmarks  of  the  greatest 
value),  and  that  give  clear  views  of  the  region  of  formation 
of  the  young  Echinoderm  (which  is  generally  opaque  in  the 
living  larva).  They  should  also  possess  sufficient  stiffness  to 
allow  of  the  larva  being  turned  about  in  any  desired  way, 
and  placed  in  any  position  under  the  microscope. 

Pluteus  larvae  should  be  fixed  in  a  cold  saturated  solution 
of  corrosive  sublimate,  in  which  they  remain  not  more  than 
two  or  three  minutes.  They  are  then  washed  with  water, 
and  brought  into  dilute  Mayer's  cochineal  (§  230).  This 
should  be  so  dilute  as  to  possess  a  barely  perceptible  tinge 
of  colour.  They  should  remain  in  it  for  from  twelve  to 
twenty-four  hours,  being  carefully  watched  the  while,  and 
removed  from  it  at  the  right  moment  and  mounted  in  balsam, 
or,  which  is  frequently  better,  in  oil  of  cloves  or  cedar- wood. 

Auricularia  and  Bipinnaria. — The  method  described  above 
is  equally  applicable  to  these  forms,  and  seems  to  be  alto- 
gether the  best  method  for  the  study  of  the  metamorphosis 
of  Bipinnaria.  The  earlier  stages  of  the  metamorphosis  of 
Auricularia  are  better  studied  by  fixing  with  osmic  acid, 
staining  with  Beale's  carmine,  and  mounting  in  glycerin. 

Larvss  of  Comatula  are  best  fixed  with  liquid  of  Lang, 
and  stained  with  dilute  borax-carmine.  It  is  important  (for 
preparations  that  are  not  destined  to  be  sectioned)  to  use 
only  dilute  borax-carmine,  as  the  strong  solution  produces 
an  over-stain  that  cannot  easily  be  reduced. 

Narcotisation  by  chloral  hydrate  before  fixing  is  useful, 
especially  for  the  study  of  Pentacrinns  larvae  and  of  the 
young  Synaptse  formed  from  Auricularia.  Without  this 
precaution  you  generally  get  preparations  of  larvae  either 
vshut  up  (Pentacrinus) ,  or  entirely  deformed  by  contraction 
(young  Synaptae). 

See  also  MACBBIDE  on  the  development  of  Amphiura  squamata,  Quart. 
Journ.  Mic.  Sci.,  xxxiv,  1892,  p.  131 ;  Journ.  Roy.  Mic.  Soc.,  1893,  p.  117 
(osmic  acid  followed  by  liquid  of  Miiller  and  alcohol;  decalcification  with 
nitric  acid  in  alcohol ;  staining  with  Mayer's  paracarmine  or  hiemalum)  ; 
and  SEELIGEE  on  the  development  of  Antedon,  Zool.Jahrb.,  Abth.  f.  Anat,, 
vi,  1892,  p.  161 ;  Zeit.  f.  wise.  Mik.,  x,  2,  1893,  p.  229. 

MACBRIDE  (Quart.  Journ.  Mic.  Sci.,  xxxviii,  1896,  p.  340)  fixes  larvre  of 


484  CHAPTER    XXXTV. 

Asterina  in  osmic  acid,  brings  into  liquid  of  Miiller  for  twelve  to  fourteen 
hours,  imbeds  in  celloidin  followed  by  paraffin  (see  §  166),  and  stains 
sections  with  carmalum  or  Delafield's  hsematoxylin,  best  after  a  foregoing 
stain  of  twenty-four  hours  in  borax  carmine. 

Coelenterata. 

866.  Thread-cells. — IWANZOFF  (Bull.  Soc.  Nat.  Moscou,  x,  1896, 
p.  97)  advises  for  the  Nematocysts  of  Actinia  maceration  by  the  HERTWIGS* 
method,  §  538,  or  better,  fixation  for  two  to  five  minutes  with  vapour  of 
osmium,  followed  by  a  short  washing  with  sea  water  or  distilled  water. 

For  Medusae  he  also  advises  the  HEETWIGS'  method,  §  538,  or  treatment 
with  a  solution  containing  methyl  green  and  gentian  violet  with  a  little 
osmic  acid. 

867.  Actinida. — Narcotisation. — For  suitable  narcotisation 
methods  see  §§  13  to  23. 

Fixation. — In  Le  At  tints,  Fauna  u.  Flora  d.  Golfes  v. 
Neapel,  ANDRES  says  that  hot  corrosive  sublimate  often  gives 
good  results.  In  the  case  of  the  larger  forms  the  solution 
should  be  injected  into  the  gastric  cavity,  and  a  further 
quantity  of  the  liquid  be  poured  over  the  animals. 

Freezing  sometimes  gives  good  results.  A  vessel  contain- 
ing Actiniae  is  put  into  a  recipient  containing  an  ice-and-salt 
freezing  mixture  and  surrounded  by  cotton- wool.  After 
freezing,  the  block  of  ice  containing  the  animals  is  thawed 
in  alcohol  or  some  other  fixing  liquid. 

See  also  Lo  BIANCO,  loc.  cit.,  p.  448. 

Maceration. — For  the  HEKTWIGS'  method  (Jen.  Zeit.,  1879, 
p.  457)  see  §  538.  The  tissues  should  be  left  to  macerate  in 
the  acetic  acid  for  at  least  a  day,  and  may  then  be  teased 
in  glycerin. 

LIST  (Zeit.  f.  wiss.  Mik.,  iv,  2,  1887,  p.  211)  treats  tentacles 
of  Anthea  cereus  and  Sagartia  parasitica  for  ten  minutes  with 
a  mixture  of  100  c.c.  of  sea  water  with  30  c.c.  of  Flemming's 
strong  liquid  (§  47),  then  washes  out  for  two  or  three  hours 
in  0*2  per  cent,  acetic  acid,  and  teases  in  dilute  glycerin. 
Picro-carmine  may  be  used  for  staining. 

868.  Zoantharia   with  Calcareous  Skeletons  are  difficult  to 
deal  with  on  account  of  the  great  contractility  of  the  polyps. 
Sublimate    solution,   which    ought  very   often   to   be   taken 
boiling,    sometimes    gives  good  results.      DE   CASTELLAKNAU 
(La   Est.   Zool.,  de  Napoles,   p.    132)   says  that  this  process 


SOME    METHODS    FOB   LOWER   ANIMALS.  485 

succeeds  well  with  Dendrophyllia,  Antipathes,  Astroides, 
Gladocora,  and  Caryophyllia. 

See  also  Lo  BIANCO,  loc.  cit.,  p.  446. 

Sections. — Besides  the  usual  methods  for  sectioning  decal- 
cified specimens,  we  have  the  methods  of  von  Koch  and 
Ehrenbaum,  §§172  and  173,  for  undecalcified  specimens. 

869.  The  Alcyonaria  have  also  extremely  contractile  polyps. 
In  a  former  edition  I  suggested  for  their  fixation  either  hot 
sublimate  solution  or  glacial  acetic  acid  (§  82).      S.  Lo  BIANCO 
has  since  recommended  essentially  similar  processes.    GARBINI 
(Manuale,  p.  151)  says  that  the  polyps  may  be  fixed  in  the 
state  of  extension  by  drenching  them  with  ether,  and  then 
bringing  them  into  strong  alcohol. 

WILSON  (Mitth.  Zool.  Stat.  Neapel,  1884,  p.  3)  kills  Alc^o- 
naria  with  a  mixture  of  one  part  of  strong  acetic  acid  and 
two  parts  of  concentrated  solution  of  corrosive  sublimate,  the 
animals  being  removed  as  soon  as  dead  and  hardened  for  two 
or  three  hours  in  concentrated  sublimate  solution. 

870.  Zoantharia  and  Alcyonaria. — BRADN  (Zool.  Anz.y  1886, 
p.  458)  proceeds  as  follows  for  Alcyonium  palmatum,   Sym- 
podium  coralloideSy  Gorgonia  verrucosa,  Caryophyllia  cyathus, 
and  Palythoa  axinellse  : — The  animals  are  left  for  a  day  or 
two    in   a   glass   vessel,    so    that    the    polyps    may   become 
thoroughly   extended.      They   are   then   suddenly    drenched 
with  a  mixture  of  20  to  25  c.c.  of  concentrated  solution  of 
sublimate  in  sea  water  with  four  to  five  drops  of  1  per  cent, 
osmic  acid.      This  is  allowed  to  act  for  five  minutes,  and  is 
followed  by  successive  alcohols. 

(This  method  also  gives  good  results  with  Hydra  and  some 
Bryozoa  and  Rotifers.) 
See  also  §  12. 

871.  Hydroidea,  Polypoid  Forms. — For  suitable  narcotisation 
methods  see  §§  13  et  seq. 

For  killing  by  heat  see  §  11. 

Fixation. — In  general  the  polyps  may  be  very  well  killed 
in  saturated  sublimate  solution,  in  which  they  should  be 
plunged  for  an  instant  merely,  and  be  brought  into  alcohol. 
The  solution  should  be  employed  cold  in  general  for  Gymno- 
blastea,  hot  for  most  Calyptoblastea. 


486  CHAPTER    XXXIV. 

Ether  attentively  administered  gives  good  results  with 
Campanularidse.  Hydra  is  very  easily  killed  by  treatment 
with  a  drop  of  osmic  acid  on  a  slide.  The  methods  for 
sections  are  the  usual  ones. 

The  methylen-blue  method  of  intra  vitam  staining  is  appli- 
cable to  this  group,  see  ZOJA,  ante,  p.  230. 

872.  Medusae  :  Fixation. — For  narcotisation  see  §  18.  There 
is  some  difficulty  in  properly  fixing  the  forms  with  contractile 
tentacles,  which  easily  roll  up  on  contact  with  reagents. 
The  best  results  I  have  had  with  these  forms  have  been 
obtained  by  means  of  VAN  BENEDEN'S  acetic  acid  method,  § 
82.  The  secret  of  success  lies  in  a  trick  of  manipulation,  due 
to  Lo  BIANCO.  Put  sufficient  acetic  acid  into  a  deepish  dish, 
hold  it  in  your  left  hand  (or,  better,  in  both  hands  if  you 
have  an  assistant),  and  keep  it  moving  in  a  circle  so  as  to 
communicate  a  vortex  motion  to  the  liquid.  Take  up  a 
medusa  in  a  spoon  with  as  little  sea  water  as  possible,  and 
throw  it  into  the  moving  liquid,  and  keep  the  liquid  steadily 
swirling  round  so  as  to  cause  the  tentacles  to  trail  out  at  full 
length  behind  the  animal  until  it  is  thoroughly  fixed,  then 
pass  carefully  into  alcohol.  Do  not,  unless  you  are  very 
expert,  try  to  fix  more  than  one  medusa  at  a  time  ;  it  is  also 
better  to  keep  the  specimens  separate,  even  in  the  alcohol, 
as,  if  several  are  together,  it  generally  happens  that  their 
tentacles  become  entangled.  Oceania  conica  and  Tiara  may 
usefully,  according  to  Lo  BIANCO,  be  narcotised  with  3  per 
cent,  alcohol  in  sea  water  before  fixation.  Liquid  of  Kleinen- 
berg,  which  I  have  seen  much  used  for  the  fixation  of  these 
and  similar  forms,  is,  in  my  opinion,  a  very  objectionable  re- 
ageiit  for  the  purpose. 

Trachymedusge  and  Acalephse  may  be  fixed  in  the  usual 
way  in  chromic  or  osmic  mixtures.  Osmic  acid  may  con- 
veniently in  some  cases  be  added  to  the  sea  water  containing 
the  animals,  which  should  be  removed  to  fresh  water  as  soon 
as  they  begin  to  turn  brown.  Cassiopeia  borbonica ,  accord- 
ing to  Lo  BIANCO,  ought  to  be  treated  with  osmic  acid  as 
described,  and  then  put  for  two  or  three  days  into  5  per 
cent,  solution  of  bichromate  of  potash.  I  have  tried  this 
process  with  good  results. 

See  further  Lo  BIANCO,  luc.  cit.}  p.  452. 


SOME    METHODS    FOR    LOWER   ANIMALS.  487 

873.  Medusae :  Sections. — I    am  not    acquainted    with   any 
perfectly  satisfactory  method  of  sectioning  these  extremely 
watery  organisms.      Paraffin  and  collodion  will  afford  good 
sections  of  some  organs,  but  are  certainly  not  satisfactory  as 
all-round  methods  for  this  group.      Some  modification  of  the 
method    employed    by    the    HEKTWIGS      (Nervensystem     der 
Medititen,  1878,  p.  5)  might  be  successful.       They  imbedded 
in   liver  with   the   aid  of  glycerin  gum,  and   hardened   the 
objects    and   the    mass   in   alcohol.      Perhaps  better  results 
might  be  obtained  by  one  of  the  freezing  methods  given  in 
§§  177  to  180. 

874.  Medusae :  Maceration. — The  methods  of  the  HERTWIGS, 
§  538,  have  deservedly  become  classical  for  the  study  of  the 
tissues  of  this  group,  especially  for  the  study  of  the  nervous 
system ;  for  ganglion  cells  and  nerve-fibrils  reduce  osmic  acid 
quicker  than  common  epithelium  cells.      Doubtless  in  many 
cases  the  pyrogallic  acid  reaction,  §361,  would  give  enhanced 
differentiation. 

The  isolation  of  the  elements  of  the  macerated  tissues  is 
best  done  by  gently  tapping  the  cover-glass  (which  may  be 
supported  on  wax  feet).  This  gives  far  better  results  than 
teasing  with  needles.  A  camel-hair  pencil  also  sometimes 
renders  good  service. 

875.  Siphonophora.— This  group  contains  some  of  the  most 
difficult  forms  to  preserve  that  are  to  be  found  in  the  whole 
range  of  the  animal  kingdom.      You  have  not  only  to  deal 
with  the  very  great  contractility  of  the  zooids,  but  with  the 
tendency  to  general  disarticulation  of  the  swimming-bells  and 
prehensile  polyps. 

The  cupric  sulphate  method  of  BEDOT  (Arch.  d.  Sci.  phys.  et 
nat.,  Juin,  1889,  t.  xxi,  p.  556),  recommended  for  the  prepara- 
tion of  Siphonophora  and  other  delicate  pelagic  animals,  is  as 
follows  : — A  large  quantity  of  15  to  20  per  cent,  solution  of 
the  salt  is  suddenly  added  to  the  sea  water  containing  the 
animals.  As  soon  as  they  are  fixed  (which  happens  in  a  few 
minutes)  a  few  drops  of  nitric  acid  are  to  be  added  and  mixed 
in  (this  is  in  order  to  prevent  the  formation  of  precipitates), 
and  the  whole  is  left  for  four  to  five  hours.  The  specimens 
are  then  to  be  hardened  before  bringing  them  into  alcohol. 


488  CHAPTER  XXXI V. 

BEDOT  recommends  that  this  be  done  with  strong  solution  of 
Flemming,  which  should  be  added  to  the  solution  of  sulphate 
containing  the  Siphonophore,  about  two  volumes  of  it  being 
taken  for  one  of  the  sulphate  solution.  The  whole  should  be 
left  for  at  least  twenty-four  hours.  Lastly,  a  few  drops  of 
25  per  cent,  alcohol  should  be  added  to  the  fluid  with  a 
pipette,  being  dropped  in  as  far  as  possible  from  the  colony, 
which  should  be  disturbed  as  little  as  possible ;  and  further 
alcohol,  of  gradually  increasing  strength,  should  be  added  so 
gradually  that  the  strength  of  70  per  cent,  be  not  attained 
under  fifteen  days  at  least.  Ninety  per  cent,  alcohol  should 
loe  used  for  definite  preservation. 

I  have  tested  this  method.  I  do  not  find  that  the  histo- 
logical  preservation  is  superior  to  that  obtained  by  means  of 
the  usual  processes ;  but  the  method  is  certainly  a  valuable 
one  in  so  far  as  it  enables  one  to  preserve  specimens  -with  all 
their  swimming-bells  and  polyps  in  situ,  a  result  which  is  not 
obtained  by  means  of  the  usual  methods. 

FRIEDLAENDER  (Biol.  Centralbl.,  x,  1890,  p.  483 ;  Journ.  Roy. 
Mic.  Soc.,  1890,  p.  804)  preserves,  this  class  of  objects  by 
inundating  them  with  a  mixture  of  1 25  parts  cupric  sulphate, 
125  parts  zinc  sulphate,  and  1000  parts  water. 

Lo  BIANCO  (loc.  cit.,  p.  454)  employs  for  the  majority  of 
Siphonophora  a  mixture  of  10  c.c.  of  saturated  solution  of 
corrosive  sublimate  with  100  c.c.  of  10  per  cent,  solution  of 
copper  sulphate.  This  is  used  as  in  BEDOT'S  process.  Diphyes, 
Rhizophysa,  and  Physaliaj  however,  are  killed  with  sublimate 
solutions ;  Velella  with  chromo-picric  acid,  or  a  mixture  of 
100  c.c.  of  sublimate  solution  with  50  c.c.  of  1  per  cent, 
chromic  acid ;  Porpita  by  poisoning  with  liquid  of  Kleinen- 
berg. 

KOROTNEFF'S  method  of  paralysing  with  chloroform  has 
been  given  in  §  15.  I  have  seen  Physophora  very  success- 
fully killed  by  the  careful  administration  of  ether. 

Preservation,  after  fixation  and  washing,  is  greatly  sim- 
plified by  the  use  of  formaldehyde  instead  of  alcohol 
(WEBER). 

DAVIDOFF  (Anat.  Anz.,  xi,  1896,  p.  505)  fixes  in  formol. 
He  gets  the  animals,  in  sea  water,  into  a  large  cylindrical 
tube  (test-tube),  plugs  its  open  end  with  cotton  wool,  and 
stands  it  up,  somewhat  sloping,  open  end  downwards,  in  a 


SOME    METHODS   FOR   LOWER  ANIMALS.  489 

vessel  half  full  of  6  to  8  per  cent,  form  oh  The  formol,  being 
lighter  than  sea  water,  diffuses  up  into  the  tube  in  about  an 
hour,  and  kills  the  animals  in  extension  and  with  little  loss 
of  the  swimming-bells ;  after  which  they  may  either  be  pre- 
served in  the  formol  itself,  or  be  further  hardened  with  other 
reagents. 

876.  Ctenophora :  Fixation. — The  small  forms  are  very  easily 
prepared  by  means  of  osmic  acid.  For  the  large  forms  see 
Lo  BIANCO,  loc.  cit.,  p.  457. 

SAMASSA  has  succeeded  in  making  sections  of  Ctenophora 
by  means  of  the  double-imbedding  method,  §  166  (see  Arch. 
/.  mik.  Anat.,  xl,  1892,  p.  157 ;  Zeit.  /.  wins.  Mik.,  1893, 
p.  340). 


Porifera. 

877.  Spongiae:  Fixation. —The  smaller  forms  can  be  fairly 
well  fixed  by  the  usual  reagents,  osmic  acid  being  one  of  the 
best.  For  the  larger  forms  no  satisfactory  fixing  agent  has 
yet  been  discovered,  so  far  as  I  can  ascertain.  The  tissues 
of  this  group  are  very  watery,  very  delicate,  very  friable  after 
hardening,  and  macerate  with  the  greatest  facility.  For  all 
but  very  small  specimens,  absolute  alcohol  is  apparently  the 
best  fixing  agent.  If  any  watery  fluid  be  preferred,  care 
should  at  all  events  be  taken  to  get  the  sponges  into  strong 
alcohol  as  soon  as  possible  after  fixation,  on  account  of  the 
rapidity  with  which  maceration  sets  in  in  watery  fluids. 
FIEDLER  (Zeit.  f.  wiss.  ZooL,  xlvii,  1888,  p.  87)  has  been  using 
(for  Spongilld),  besides  absolute  alcohol,  an  alcoholic  subli- 
mate solution  and  the  liquids  of  Kleinenberg  and  Flemming 
with  good  effect. 

Staining. — On  account  of  the  great  tendency  to  maceration, 
I  hold  that  alcoholic  stains  should  be  alone  employed  for 
staining  sponges,  and  I  particularly  recommend  Mayer's  tinc- 
ture of  cochineal,  §  230.  VON  LENDKNFELD  (Zeit.f.  wiss.  Mik., 
xi,  1,  1894,  p.  22)  uses  aqueous  solutions  of  Congo  red  and 
&nilin  blue  for  the  coloration  of  collar-cells. 

Sectioning. — Calcareous  sponges  maybe  decalcified  in  alcohol 
slightly  acidified  with  hydrochloric  acid,  and  then  imbedded 


490  CHAPTER    XXXIV. 

in  the  usual  way.  Siliceous  sponges  may  be  desilicified  by 
Mayer's  hydrofluoric  acid  method  (see  §  574). 

For  ROUSSEAU'S  methods  see  §§  558,  574.  VOSMAER  and. 
PEKELHARING  decalcify  with  a  solution  of  picric  acid  in  abso- 
lute alcohol  (see  Zeit.  f.  u-iss.  Mik.,  xv,  1899,  p.  462). 

See  also  Johnstone-Lavis  and  Vosmaer,  §  174. 

Preparation  of  Hard  Parts. — Siliceous  spicules  are  easily 
cleaned  for  mounting  by  treating  them  on  a  slide  with  hot 
concentrated  nitric  or  hydrochloric  acid,  or  solution  of  potash 
or  soda.  The  acids  mentioned  are  very  efficient,  but  it  must 
be  pointed  out  that  they  will  attack  the  silex  of  some  delicate 
spicules.  Thus  DEZSO  found  that  the  small  stellate  spicules 
of  the  cortex  of  Tethya  lyncurium  are  completely  dissolved 
by  boiling  hydrochloric  acid.  Potash  solution  is,  therefore, 
frequently  to  be  preferred,  notwithstanding  that,  in  my  ex- 
perience, it  does  not  give  such  clean  preparations. 

According  to  NOLL,  eau  de  Javelle  is  preferable  to  any  of 
these  reagents  (see  §  555). 

Impregnation  with  Silver  (see  §  343) . 

Embryos  and  Larvae. — MAAS  (Zool.  Jahrb.,  Abth.  Morph., 
vii,  1894,  p.  334)  fixes  larvae  in  liquid  of  Flemming  or  Her- 
mann, one  to  three  minutes,  and  stains  with  borax-carmine, 
or  with  gentian  violet  and  Orange  Gr  (Flemming). 

DELAGE  (Arch.  Zool.  Exper.,  x,  1892,  p.  421)  fixes  larvae  of 
Sponyilla  that  have  settled  down  on  cover-glasses  for  three 
minutes  in  absolute  alcohol,  stains  in  alcoholic  carmine,  §  229, 
and  brings  through  alcohol  into  oil  of  bergamot,  then  either 
mounts  direct  in  balsam,  or  detaches  the  larvae  from  the 
cover  and  imbeds  in  paraffin  (three  minutes). 


Protozoa. 

878.  Introductory. — Since  the  Protozoa  may  be  considered 
as  free  cells,  it  is  evident  that  the  reagents  and  methods  of 
cytology  are  in  great  part  applicable  to  this  group.  One  of 
the  most  generally  useful  of  these  reagents  will  be  found  in 
the  acid  solution  of  methyl  green  ;  it  is  the  reagent  that  allows 
of  the  readiest  and  best  demonstration  of  the  presence  and 


SOME    METHODS    FOB    LOWER    ANIMALS.  491 

form  of  the  nucleus  and  nucleolus  (BALBIANI  et  HKKNEGUY, 
Cornpt.  rend.  Sue.  de  BioL,  1881,  p.  131). 

Amongst  useful  reagents  not  mentioned  in  the  following 
descriptions  of  the  methods  employed  by  different  authors,  I 
call  attention  to  the  Aveak  solutions  of  alum,  potash,  and  borax, 
which  serve  to  demonstrate  the  striations  of  the  cuticle,  and 
the  insertions  of  the  cilia  of  Infusoria. 

See  also  MAGGI,  Technica  protistologica,  Milano,  1895. 

879.  Methods  for  quieting  Infusoria. — The  narcotisation 
methods,  §§  18  to  22,  are  available  for*  this  purpose. 

According  to  SCHURMA.YER  (Jen.  Zeit.,  xxiv,  1890,  pp.  402 — 
470;  Zeit.  f.  u'iss.  Hik.,  vii,  4,  1891,  p.  493)  nitrate  of 
strychnin  in  weak  solution,  0*01  per  cent,  or  less,  gives  good 
results  with  some  forms,  amongst  which  are  Stentor  and 
Carchesium.  Antipyrin  in  concentrated  solution  (O'l  per 
cent.),  or  cocaine  of  0*01  per  cent.,  seems  only  to  have  given 
good  results  as  regards  the  extension  of  the  stalk  in  stalked 
forms. 

EISMOND  (Zool.  Anz.,  xiii,  1890,  p.  723)  has  proposed  a 
mechanical  means  of  slowing  the  movements  of  small 
organisms  (small  worms  and  Crustacea  as  well  as  Ciliata). 
He  directs  that  a  drop  of  thick  aqueous  solution  of  cherry- 
tree  gum  be  added  to  the  water  containing  the  organisms 
(gum  arabic  and  the  like,  it  is  stated,  will  not  do).  The 
objects  remain  fixed  in  their  places,  with  cilia  actively  moving, 
and  all  vital  processes  retaining  their  full  activity. 

CERTES  (Bull.  Soc.  Zool.  France,  xvi,  1891,  p.  93;  Journ. 
Boy.  Mic.  Soc.,  1891,  p.  828)  has  found  that  the  method  gives 
excellent  results.  He  has  also  found  that  an  intra  vitam 
sta-in  may  be  obtained  by  adding  methyl  blue  or  "  violet 
dahlia,  No.  170,"  to  the  gum  solution. 

A  similar  process  has  been  worked  out  by  JENSEN  (after 
STAHL  ;  see  BioL  GentralU.,  xii,  1892,  18,  19,  p.  556  ;  Zeit.  f. 
wit*.  Mik.,  ix,  4,  1893,  p.  483;  Journ.  Roy.  Mic.  Soc.,  1892, 
p.  891).  A  solution  of  3  grammes  of  gelatin  in  100  c.c.  of 
ordinary  water  is  made  by  the  aid  of  heat.  This  makes  a 
jelly  at  the  normal  temperature.  It  is  slightly  warmed,  and 
a  drop  of  it  is  mixed  in  a  watch  glass  with  a  drop  of  water 
containing  the  organisms.  This  plan  is  said  to  afford  great 
facilities  for  the  vivisection  of  Infusoria. 


492  CHAPTER    XXXIV. 

880.  Staining  intra  vitam. — The  possibility  of  staining  In- 
fusoria intra  vitam  was  discovered  independently  by  BEANDT 
(Verh.  d.  physiol.  Ges.  Berlin,  1878),  by  CEETES  (Bull.  Soc. 
ZooL,  25  janv.,  1881),  and  by  HENNEGUY  (Soc.  Philom.,  12 
fev.,  1881).  See  on  this  subject  §  201. 

BEANDT  recommends  a  1  :  3000  solution  of  Bismarck  brown; 
also  (Biol.  CentralbL,  i,  1881,  p.  202)  "a  dilute  solution  of 
hasmatoxylin." 

CEETES  (op.  tit.,  pp.  21,  226,  264,  and  ZooL  Anz.,  iv,  1881, 
pp.  208,  287)  found  that  living  Infusoria  stain  in  weak  solu- 
tions of  cyanin,  Bismarck  brown,  dahlia,  violet  5  B,  chrysoidin, 
nigrosin,  methylen  blue,  malachite  green,  iodine  green,  and 
other  tar  colours,  and  haematoxylin.  The  solutions  should  be 
made  with  the  liquid  that  constitutes  the  natural  habitat  of 
the  organisms.  They  should  be  very  weak,  that  is  of 
strengths  varying  between  1  :  10,000  and  1  :  100,000.  For 
cyanin,  1  :  500,000  is  strong  enough. 

As  to  the  staining  of  the  Nucleus,  see  PRZESMYCKI,  Biol. 
CentralbL,  vii,  1897,  p.  321  ;  and  as  to  that  of  the  Granida, 
the  same  author,  Zeit.  f.  wiss.  Mik.,  xiii,  1896,  p.  478. 

Examination  in  a  coloured  medium  in  which  the  organisms 
do  not  stain,  but  show  up  on  a  coloured  background  in  a 
manner  that  produces  somewhat  the  effect  of  dark-ground 
illumination,  is  sometimes  helpful.  CEETES  (Bull.  Soc.  ZooL 
de  France,  xiii,  1888,  p.  230)  recommends  solution  of  anilin 
black  for  this  purpose ;  Infusoria  will  live  in  it  for  weeks. 
FABEE-DOMEKGUE  (Ann.  de  Microgr.,  ii,  1889,  p.  545;  Journ. 
Roy.  Hie.  Soc.,  1889,  p.  832)  recommends  concentrated  solu- 
tion of  diphenylamin  blue. 


881.  Fixing  and  Preserving. — For  killing  by  heat  see  §  11, 
p.  12. 

PFITZNEB  (Morph.  Jahrb.,  xi,  1885,  p.  454)  used  concen- 
trated solution  of  picric  acid  run  in  under  the  cover. 

BLANC  (ZooL  Anz.,  vi,  1882,  p.  22)  advises  liquid  of  Kleinen- 
berg  diluted  with  about  a  volume  of  water,  and  acidified  with 
acetic  acid. 

G-EZA  ENTZ  (ibid.,  iv,  1881,  p.  575)  adds  it  to  the  water 
containing  the  organisms  in  a  ivatch-glass. 

KOESCHELT  (ibid.,  v,  1882,  p.  217)  employs    in   the   same 


SOME    METHODS    FOK    LOWER    ANIMALS.  493 

way  1   per  cent,  osmic   acid,   or,  for  Amoebae,  2  per    cent, 
chromic  acid. 

LANSBERG  (ibid.,  p.  336)  advises  the  same  reagents,  but 
recommends  bringing  the  organisms  into  the  fixing  liquid 
with  a  pipette. 

For  fixation  with  iodine  (KENT)  or  iodine  vapour  (OVERTON), 
see  §  88. 

For  sulphurous  acid,  §  65. 

CATTANEO  (Bollettino  Scientifico,  iii  and  iv;  Journ.  Roy.. 
Mic.  Soc.,  1885,  p.  538)  recommends  fixing  for  a  few  minutes 
with  -j-  per  cent,  solution  of  chloride  of  palladium,  which 
hardens  in  a  few  minutes  without  blackening. 

BRASS  (Zeit.  f.  wiss.  Mile.,  i,  1884,  p.  39)  employs  the  follow-, 
ing  liquid  : 

Chromic  acid        .  .  .  .1  part. 

Platinum  chloride          .  .  .      1      „ 

Acetic  acid  .  .  .  .      1      „ 

Water          .  .  .400  to  1000  parts. 

CERTES  (Comptes  rend.,  Ixxxviii,  1879,  p.  433)  fixes  with  2  per  cent. 
isi'c)  osmic  acid,  or  its  vapours  (10  to  30  minutes).  For  details  see 
previous  editions. 

Dr  PLESSIS  (Vooi  et  YUNG,  Traite  Anat.  Comp.  Prat.,  p.  92)  recom- 
mends fixation  with  0'2  per  cent,  solution  of  corrosive  sublimate.  Let  the 
preparation  dry  up,  and  if  the  organisms  have  preserved  their  shape,  stain 
;md  mount  in  balsam.  This  seemingly  barbarous  mode  of  procedure  is 
said  to  give  very  fine  preparations  when  successful. 

FOL  (Lehrb.,  p.  102)  fixes  delicate  marine  Infusoria  (Tin- 
tinnodea)  with  the  perchloride  of  iron  solution  (§  80),  added 
to  the  water  containing  them,  and  stains  with  gallic  acid  as 
directed,  §  362,  and  states  that  this  is  the  only  method  that 
has  given  him  good  results,  especially  as  regards  the  preser- 
vation of  cilia. 

Lo  BIANCO  (loc.  cit.,  p.  444)  fixes  Gregarinae  with  picro-sul- 
phuric  acid  (one  hour),  Yorticellae  with  hot  sublimate, 
Acinetae  with  sublimate  in  sea  water,  or  with  osmic  acid, 
Thalassicola  with  0'5  per  cent,  chromic  acid  (one  hour), 
AcanthometrBe  and  Aulacanthae  with  50  per  cent,  alcohol  or 
with  concentrated  sublimate,  or  by  adding  a  little  osmic  acid 
to  the  water.  For  Sphaerozoa  he  proceeds  as  BRANDT,  below. 

ZOGRAF  fixes  Rhizopoda  and  Infusoria  as  Rotatoria,  §  853, 
but  without  narcotisation. 


494  CHAPTER  XXXIV. 

BRANDT  (Fauna  u.  Flora  Golf.  Neapel,xiii,  1885, p.  7;  Journ. 
Roy.  Hie.  Soc.,  1888,  p.  665)  fixes  Sphserozoa,  according  to  the 
species,  either  with  chromic  acid  of  0'5  per  cent,  to  1  per 
cent,  (half  an  hour  to  an  hour),  or  with  a  mixture  of  equal 
volumes  of  sea  water  and  70  per  cent,  alcohol  with  a  little 
tincture  of  iodine  for  a  quarter  to  half  an  hour,  or  with  a 
5  to  15  per  cent,  solution  of  sublimate  in  sea  water. 

Sporozoa. — WASIELEWSKI  (Sporozoenkunde,  Jena,  1896,  p. 
153)  lays  great  stress  on  the  study  of  the  living  organisms, 
either  in  their  natural  medium,  or  in  normal  salt  solution,  or 
in  a  medium  composed  of  20  parts  white  of  egg,  200  of  water, 
and  1  of  common  salt.  He  fixes  Gregaringe  and  Coccidia 
with  osmic  acid,  sublimate,  or  picro-sulphuric  acid,  and 
Myxosporidia  with  liquid  of  Flemming.  He  stains  G-regarinae 
with  safranin,  picro-carmine,  etc.,  besides  employing  gold 
•chloride,  silver  nitrate,  acetic  acid,  ammonia,  etc.,  and 
Myxosporidia  with  safranin  or  gentian  and  eosin. 

See  also  the  methods  of  FABRE-DOMERGUE,  Ann.  de  Microgr.,  ii,  1889, 
p.  545,  and  1890,  p.  50 ;  SCHEWIAKOFF,  Biblioth.  ZooL,  v,  1889,  p.  5  ; 
Journ.  Roy.  Hie.  Soc.,  1889,  pp.  832,  833 ;  ZOJA,  Boll.  Sci.  Pavia,  1892 ; 
Zeit.  f.  wiss.  Mik.,  ix,  4,  1893,  p.  485 ;  LONGHI,  Bull.  Mus.  Zool.  Univ. 
Genova,  4,  1892;  Zeit.  f.  wiss.  Mik.,  ix,  4,  1893,  p.  483;  LAUTERBORN, 
Zeit.  wiss.  Zool.,  lix,  1895,  p.  170;  SCHAUDINN,  ibid.,  p.  193;  BALBIANI, 
Zool.  Anz.,  xiii,  1890,  p.  133;  KARAWAIEW,  ibid.,  xviii,  1895,  p.  286. 

882.  Sections. — Sections  of  the  larger  Protozoa,  and 
amongst  them  of  the  larger  forms  of  Infusoria  (Stentor, 
Bursaria,  Nyctotherus),  may  be  obtained  without  much  diffi- 
culty. The  organisms  should  be  strongly  fixed,  then  dehy- 
drated and  cleared,  and  brought  into  melted  paraffin  in  a 
small  watch  glass.  After  a  few  minutes  therein  they  are 
brought  on  a  cataract  needle  on  to  a  small  block  of  paraffin, 
and  arranged  there  with  a  heated  needle  (p.  93)  and  sec- 
tioned. They  may  be  stained  after  fixation,  or  the  sections 
may  be  stained  on  the  slide,  §  182  or  183. 

LAUTEKBORN  (loc.  cit.  last  §)  brings  the  objects  through 
chloroform  into  paraffin  in  a  small  glass  tube,  and  after 
cooling  breaks  the  tube  and  so  obtains  a  cylinder  of  paraffin 
with  the  objects  ready  for  cutting, 

HOYER  (Arch.  mik.  Anat.,  liv,  1899,  p.  95)  performs  all 
the  operations  in  a  glass  cylinder  (5  cm.  long  and  7 'mm. 
wide)  open  at  both  ends,  but  having  a  piece  of  moist  parch- 


SOME    METHODS    FOE    LOWER  ANIMALS.  495 

ment  paper  tied  over  one  of  the  openings.  It  is  then  not 
necessary  to  break  the  cylinder  ;  by  removing  the  parchment 
paper  the  paraffin  can  be  pushed  out  of  it  in  the  shape  of  a 
cylinder  containing  the  objects  imbedded  at  one  end  of  it. 

See  also  the  watch  glass  method,  p.  94,  and  the  papers 
there  quoted;  also  PRZESMYCKJ,  loc.  cit.,  §  880. 

883.  Demonstration  of  Cilia   (WADDINGTON,  Journ.  Roy.  Mic. 
Soc.,  1883,  p.  185). — A  drop  of  solution  of  tannin,  or  a  trace 
of  alcoholic  solution  of  sulphurous  acid,  added  to  the  water 
containing  the  living  organisms. 

884.  Stains  for  Flagella. — The  method   of    LOFFLER  has  run 
through  several  forms  (Centralbl.f.  BacterioL,vi,  1889,  p.  209  ; 
vii,  1890,  p.  625;  Zeit.  f.  wiss.  Nik.,  vi,  3,  1889,  p.  359;  vii, 
3,  1890,  p.  368;  Journ.  Roy.  Mic.  Soc.,  1889,  p.  711;   1890, 
p.  678),  of  which  that  given  here  is  the  latest.      To   10  c.c. 
of  a  20  per  cent,  solution  of  tannin  are  added  5  c.c.  of  cold 
saturated  solution  of  ferrous  sulphate  and   1   c.c.  of  (either 
aqueous  or  alcoholic)    solution  of  fuchsin,  methyl  violet,  or 
"  Wollschwarz."      (The  mixture  will  require  for  some  forms 
the  addition  of  a  few  drops  of  1  per  cent,  solution  of  caustic 
soda ;  e.g.  for  typhoid  bacilli,  1  c.c. ;  for  Bacillus  subtilis,  28 
to  30  drops ;  for  bacilli  of  malignant  oedema,  36  to  37  drops. 
Some  other  forms  will  require  besides  the  addition  of  a  trace 
of  sulphuric  acid  to  the  soda  solution, — so  for  cholera  bacteria, 
half  a  drop  to  1  drop;   for  Spirillum  rubrum,  9  drops). 

Cover-glass  preparations  are  made  and  fixed  in  a  flame  in 
the  usual  way,  special  care  being  taken  not  to  over-heat. 
Whilst  still  warm,  the  preparation  is  treated  with  mordant 
(i.  e.  the  above-described  mixture),  and  is  heated  for  half  a 
minute,  until  the  liquid  begins  to  vaporise,  after  which  it  is 
washed  in  distilled  water  and  then  in  alcohol.  It  is  then 
treated  in  a  similar  manner  with  the  stain,  which  consists  of 
a  saturated  solution  of  fuchsin  in  anilin  water,  the  solution 
being  preferably  neutralised  to  the  point  of  precipitation  In- 
cautious addition  of  O'l  per  cent,  soda  solution. 

BCNGE  (Journ.  Roy.  Mic.  Soc.,  1894,  p.  640;  Zeit.  f. 
Mik.j  xiii,  1896,  p.   96)  makes  the  mordant  by  mixing  three 
parts  of  the  tannin  solution   with    1  of  Liquor  Ferri  Sexfjui- 
chlorati  diluted  twentyfold  with  water,  and  lets  the  mixture 


496  CHAPTER  XXXIV. 

ripen  for  some  days  exposed  to  the  air,  or  (Journ.,  1895, 
pp.  129,  248)  adds  to  it  a  few  drops  of  hydrogen  peroxide, 
until  it  becomes  red-brown,  when  it  is  shaken  up  and  filtered 
on  to  the  cover-glass  and  allowed  to  act  for  a  minute.  The 
cover-glass  is  then  mopped  up  and  dried,  and  stained  with 
carbol-gentian. 

VAN  ERMENGEM  (Journ.  1894,  p.  405)  fixes  for  a  few  minutes 
with  a  mixture  of  1  part  2  per  cent,  osmic  acid,  and  2  parts 
10  to  25  per  cent,  solution  of  tannin,  washes,  treats  with  0*25 
to  0'5  per  cent,  solution  of  nitrate  of  silver,  then  for  a  few 
seconds  with  a  mixture  of  5  parts  gallic  acid,  3  of  tannin,  10 
of  acetate  of  soda,  and  330  of  water,  then  puts  back  again 
into  the  silver  for  a  short  time,  then  washes  and  mounts. 

See  also  the  modifications  of  this  method  by  STEPHENS,  ibid., 
1898,  p.  685,  and  Gordon,  ibid.,  1899,  p.  235,  and  the 
methods  of  TRENKMANN  (Centralbl.,  vi,  1889,  p.  433;  Zeit.  f. 
wiss.  Mik.,  vii,  1,  1890,  p.  79);  BROWN  (Journ.  Roy.  Mic. 
Soc.,  1893,  p.  268)  ;  JDLIEN  (ibid.,  1894,  p.  403)  ;  SCLAVO  (Zeit. 
f.  wiss.  Mik.,  xiii,  1896,  p.  96)  ;  HESSERT  (ibid.,  p.  96)  ;  Mum 
(Journ.  Roy.  Mic.  Soc.,  1899,  p.  235)  ;  McCRORiE  (ibid.,  1897, 
p.  251 ;  he  stains  for  two  minutes  in  a  mixture  of  equal  parts 
of  concentrated  solution  of  night-blue,  10  per  cent,  solution 
of  alum,  and  10  per  cent,  solution  of  tannic  acid)  ;  ZETTNOW 
(ibid.,  1899,  pp.  662,664);  MORTON  (ibid.,  1900,  p.  131); 
WELCKE  (ibid.,  p.  132). 


APPENDIX. 


885.  The  Usual  Alcohols. — The  following,  or  a  similarly 
spaced  series  of  alcohols,  should  be  kept  on  the  table. 

Absolute  Alcohol— See  §  101.  The  so-called  "absolute 
alcohol "  of  commerce  is  generally  of  about  98  per  cent, 
strength.  This  grade  is  convenient,  but  not  necessary  for 
ordinary  work.  Water  in  alcohol  may  be  detected  (YvoN, 
Compf.es  Rend.,  cxxv,  1897,  p.  1181)  by  adding  a  little 
coarsely  powdered  carbide  of  calcium;  the  merest  trace  of 
water  causes  a  disengagement  of  acetylene  gas,  and  on 
agitation  the  alcohol  becomes  turbid  with  calcium  hydrate. 

95  per  cent.  Alcohol. — This  is  the  average  strength  of  the 
common  strong  commercial  alcohol,  which  ranges  in  general 
from  94  per  cent,  to  96  per  cent,  according  to  temperature. 
The  strength  of  this,  or  of  the  following,  should  be  deter- 
mined by  means  of  an  areometer  (Gay  Lussac's  being  very 
convenient),  so  as  to  form  a  starting-point  for  the  following 
mixtures,  which  may  be  made  by  means  of  the  table,  next  §. 
This  is  the  usual  grade  for  dehydrating  before  clearing.  It 
is  the  highest  grade  that  should  be  used  for  dehydrating 
celloidin  sections. 

90  per  cent.  Alcohol. — May  be  made  by  taking  100  vols. 
95  per  cent,  alcohol  and  5*5  vols.  water.  Oil  of  bergamot 
will  clear  from  this  grade.  This  is  the  usual  strength  of  the 
strongest  commercial  methylated  spirit,  which,  if  free  from 
mineral  naphtha,  is  used  by  some  persons  instead  of  pure 
alcohol.  A  writer  in  the  Athenseum,  June  4th,  1898,  p.  728, 
says,  "  We  believe  that  all  ordinary  methylated  spirit 

32 


498  APPENDIX. 

dealt  with  by  retailers  now  contains,  by  law,  at  least  %  per 
cent,  of  petroleum,  and  that  methylated  spirit  free  from  it 
can  only  be  obtained  by  taking  special  measures."  If  this 
be  so,  I  should  say  it  ought  not  to  be  used  at  all.  If 
naphtha  be  present  in  alcohol  it  will  become  turbid  on  addi- 
tion of  water. 

85  per  cent.  Alcohol. — Rectified  spirit,  B.  P.,  is  a  little 
weaker  than  this,  viz.  84*5  per  cent. 

70  per  cent.  Alcohol. — Only  exceptionally  powerful  clearers, 
such  as  anilin  oil,  will  clear  from  this  grade:  see  §  121. 
This  is  the  proper  grade  for  the  temporary  preservation  of 
tissues  intended  for  histological  study  (but  see  the  remarks 
on  preservation  on  page  5) ;  higher  grades  are  best  not  taken 
unless  it  is  desired  to  harden.  This  is  the  proper  grade  for 
washing  out  borax-carmine  stains,  sublimate  after  fixing,  etc. 

50  per  cent.  Alcohol. — This  is  the  strength  of  proof  spirit. 

"  One-third  Alcohol."— See  §  100. 

886.  Table  for  diluting  Alcohol  (after  GAY-LUSSAC). — To  use 
this  table,  find  in  the  upper  horizontal  row  of  figures  the 
percentage  of  the  alcohol  that  it  is  desired  to  dilute,  and  in 
the  vertical  row  to  the  left  the  percentage  of  the  alcohol  it 
is  desired  to  arrive  at.  Then  follow  out  the  vertical  and 
horizontal  rows  headed  respectively  by  these  figures,  and  the 
figure  printed  at  the  point  of  intersection  of  the  two  rows  will 
show  how  many  volumes  of  water  must  be  taken  to  reduce 
one  hundred  volumes  of  the  original  alcohol  to  the  required 
grade.  Thus,  if  it  be  required  to  manufacture  some  70  per 
cent,  alcohol,  starting  with  90  per  cent.,  we  find  the  figure 
90  in  the  upper  column,  the  figure  70  in  the  vertical  column, 
and- at  the  point  of  intersection  we  read  31*05,  showing  that 
a  fraction  more  than  31  volumes  of  water  must  be  added  to 
100  volumes  of  90  per  cent,  alcohol.  Or  similarly,  if  we 
wish  as  before  to  make  70  per  cent,  alcohol,  but  start  with 
an  alcohol  of  85  per  cent.,  we  find  that  23*14  volumes  of 
water  must  be  employed. 


APPENDIX. 


499 


Weaker  grade 
required. 

ORIGINAL  GHADK. 

90 

p.  100. 

85 

p.  100. 

80 

p.  100. 

75 

p.  100. 

70 

p.  100. 

65    |%) 

p.  100.     p.  100. 

55       50 

p.  100.     p.  100. 

P 

100. 

85 

6-56 

80 

1379 

6-83 

75 

21-89 

14-48 

7-20 

1 

70 

31-05 

23-14 

15-35 

7-64 

65 

41-53 

3303 

24-66 

16-37 

8-15 

60 

53-65 

44-48 

35-44 

26-47 

17-58 

8-76 

55* 

67-87 

57-90 

48-07 

38-32 

28-63 

19-02      9-47 

. 

50 

84-71 

73-90 

63-04 

52-43 

41-73 

31-25    20-47 

1035 

45 

105-34 

93-30 

81-38 

69-54 

57-78 

46-09    34-46  !  22-90    11-41 

40 

130-80 

117-34 

104-01 

9076 

77-58 

64-48    51-43 

38-46    25-55 

35 

163-28 

148-01 

132-88 

117-82 

102-84 

87-93 

73-08    58-31    43-59 

30 

206-22 

188-57 

171-05 

153-61 

136-04 

118-94101-71    84-54    67'45 

887.  Chemicals,   Stains,   and    Apparatus. — Addresses    from 
which  it  is  recommended  that  these  be  obtained  are  given 
in  §  204. 

888.  Cleaning  Slides  and  Covers. — New  ones  may  be  soaked 
at  once  in  one  of  the  following  solutions,  washed  with  water 
and  alcohol,  and  dried  with  a  cloth. 

For  used  ones,  if  a  balsam  mount,  warm,  push  the  cover 
into  a  vessel  with  xylol  or  the  like,  and  put  the  slide  into 
another  vessel  with  the  same,  leave  for  a  few  days,  and  then 
put  into  strong  alcohol. 

See  also  p.  142. 

HENEAGE  GIBBES,  Journ.  Roy.  Mic.  Soc.,  iii,  1880,  p.  392. — 
Place  the  cover-glasses  in  strong  sulphuric  acid  for  an  hour 
or  two,  wash  well  until  the  drainings  give  no  acid  reaction  ; 
wash  first  with  methylated  spirit,  and  then  with  absolute 
alcohol,  and  wipe  carefully  with  an  old  silk  handkerchief . 

SEILER,  ibid.,  p.  508. — New  slides  and  ftvers  are  placed 
for  a  few  hours  in  the  following  solution  : 


500  APPENDIX. 

Bichromate  of  potash      ...        3  ounces.  3.t 

Sulphuric  acid       .  .  .          .3  fluid  ounces.     ^>; 

Water    .    £ 25          „ 

Wash  with  water.  The  slides  may  be  simply  drained  dry  ; 
the  covers  may  be  wiped  dry  with  a  linen  rag. 

Slides  and  covers  that  have  been  used  for  mounting  either 
with  balsam  or  a  water  medium  are  treated  as  follows  : — The^ 
covers  are  pushed  into  a  mixture  of  equal  parts  of  alcohol 
and  hydrochloric  acid,  and  after  a  few  days  are  put  into  the 
bichromate  solution  and  treated  like  new  ones.  The  slides 
are  scraped  free  of  the  mounting  medium  with  a  knife  and 
put  directly  into  the  bichromate  solution. 

FOL  (Lehrb.,  p.  132)  recommends  either  a  solution  con- 
taining 3  parts  of  bichromate,  3  of  sulphuric  acid,  *nd  40  of 
water ;  or  simply  dilute  nitric  acid. 

G-ARBINI  (Manuals,  p.  31)  puts  slides  for  a  day  into  10  per 
cent,  sulphuric  acid,  then  washes,  first  with  water  and  then 
with  alcohol. 

BEHEENS  (Zeit.  f.  wiss.  Mik.,  1885,  p.  55)  treats  slides 
first  with  concentrated  nitric  acid,  then  with  water,  alcohol, 
and  ether. 

JAMES  (Journ.  Roy.  Mic.  Soc.,  1886,  p.  548)  treats  used 
slides  with  a  mixture  of  equal  parts  of  benzin,  spirit  of 
turpentine,  and  alcohol. 

KNAUER  (Centralbl.  f.  Bakt.,  x,  1891,  p.  8;  Zeit.  f.  wiss. 
Mik.,  ix,  2,  1892,  p.  187 ;  Journ.  Roy.  Mic.  Soc.,  1891,  p.  833) 
recommends  boiling  for  twenty  or  thirty  minutes  in  10  per 
cent,  lysol  solution,  then  rinsing  with  cold  tap  water  till 
clear. 

NIAS  (Journ.,  pag.  cit.)  finds  it  is  sufficient  to  boil  with 
washing  soda,  and  rinse. 

ZIELINA  (Zeit.  f.  wiss.  Mik.,  xiv,  1897,  p.  368)  puts  used 
slides  for  some  days  into  water,  and  scrapes  off  the  balsam 
with  a  piece  of  wood.  He  treats  new  slides  or  covers  for  a 
few  minutes  with  glacial  acetic  acid,  washes,  and  dries  with 
a  cloth. 

889.  Re-staining  Old  Mounts  (HENNEGUY,  from  the  last  edition 
of  the  Traitedes  Methodes  techniques  de  I'Anat.  microsco pique, 
LEE  et  HENNEOTY). — It  is  probably  not  generally  known  that 
balsam  mounts  the  stain  of  which  has  faded,  or  which  it  may 


APPENDIX.  501 

be  desired  to  submit  to  some  other  staining  process  or  mount 
in  some  other  medium,  may  often  with  great  advantage  be 
re- stained  and  re-mounted.  All  that  is  necessary  is  to  put 
the  slide  into  a  tube  of  xylol  or  benzol  till  the  cover  falls  off 
(about  two  days),  wash  well  for  some  hours  in  clean  xylol, 
and  pass  through  alcohol  into  the  new  stain.  Since  this  was 
pointed  out  to  me  by  Dr.  Henneguy  I  have  unmounted  and 
re-stained  a  considerable  number  of  old  preparations,  some 
of  them  over  fifteen  years  old,  and  have  been  most  agree- 
ably surprised  at  the  results  obtained.  I  have  succeeded  in 
every  case  with  series  of  sections  mounted  on  Mayer's  albu- 
men, or  by  the  water  method.  For  shellac  mounted  series 
E.  MEYER  (BioL  Centralb.,  x,  1890,  p.  509)  removes  the 
covers  and  the  balsam  with  chloroform,  pours  quickly  a 
2  per  cent,  solution  of  photoxylin  or  celloidin  over  the  slide, 
and  after  a  few  seconds  brings  it  first  into  70  per  cent, 
alcohol,  then  into  90  per  cent.,  which  dissolves  the  shellac, 
then  removes  the  membrane  of  collodion  with  the  sections 
under  70  per  cent,  alcohol,  and  stains. 

890.  Gum  for  Labels. — Labels  stuck  on  glass  often  strip  off. 
This  may  be  avoided   (MARPMANN,   Zeit.  f.  Angew.  Mile.,  ii, 
1896,  p.  151  ;  Journ.  Roy.  Mic.  Soc.,  1897,  p.  84)  by  means 
of  the  following  adhesives  :   120  grammes  of  gum  arabic  are 
dissolved  in  a  quarter  of  a  litre  of  water,  and  30  grammes  of 
gum  tragacanth  in  a  similar  quantity.      After  a  few  hours 
the  tragacanth  solution  is  shaken  until  it  froths,  and  mixed 
with  the  gum  arabic  solution.     Strain  through  linen  and  add 
150  grammes  of  glycerin  previously  mixed  with  2  4  grammes 
of  oil  of  thyme. 

For  other  receipts  see  previous  editions. 

891.  Orientation   (Addendum  to   §§  133,  134,  588). — For 
further  details  as  to  the  BORN-PETER  method  of  establishing 
definition  lines  on  paraffin  blocks,  see  Verh.  d.  Anat.  Ges., 
xiii  Vers.,  1899  (Jena,  Fischer),  p.  134. 

892.  Knife-heating  Apparatus  (Addendum  to  §  135,  p.  110)  .- 
An  apparatus  for  warming  or  cooling  both  the  knife  and  the 
paraffin  whilst   cutting  is  described  by  HELD  in  Arch.  Anat. 
Phytt.,  Anat.  Abth.,  1897,  p.  345. 


502  APPENDIX. 

893,  Iron  Hsematoxylin  (Addendum  to  §  255,  p.  191)  .—HELD 
(op.  cU.t  SuppL,  p.  158)  finds  it  an  improvement  to  add  to 
the  staining  bath  a  very  little  of  the  iron-alum  solution  until 
a  scarcely  perceptible  precipitate  is  produced. 

894.  Neuro-fibrils    (Addendum    to    §    707).— BETHE   (Zeit. 
f.  wiss.  Mik.,  xvii,   1,   1900,  pp.  13 — 35)   has  now  minutely 
described  the  method  mentioned  on  p.  395.    Pieces  of  central 
nervous  system  are  fixed  for  twenty-four  hours  in  nitric  acid 
of  from  3  per  cent,  to  7*5  per  cent,   strength,  and  brought 
direct  into  alcohol  of  96  per  cent,  for  a  day  or  more.      They 
are  put  for  twelve  to  twenty -four  hours  into  a  mixture  of  one 
part  of  ammonia   (of  sp.  gr.  0*95)   with  three   of  water  and 
eight  of  96  per  cent,  alcohol,  then  for  six  to  twelve  hour's 
into  a  mixture  of  one  part  concentrated  nitric  acid  with  three 
of  water  and  eight  to  twelve  of   alcohol,   then    for   ten   to 
twelve  into  pure  alcohol,  and  thence  for  not  more  than  two 
to  six  hours  into  water.      They  are  now  mordanted  for  twenty- 
four  hours  in  a  4  per  cent,  solution  of  ammonium  molybdate, 
brought  for  twenty-four  hours  into  alcohol,   and  imbedded 
in  paraffin  (not  celloidin).    Sections  are  seriated  on  albumen 
and  "  differentiated  " — by  which  the  author  means  washed 
out — with   water.      About    1   to    1*5    c.c.   of   distilled  water 
should  be  poured  on  to  the  slide  so    as  to  form    over   the 
sections  a  layer  1*5  to  2  mm.  deep,  and  the  slide  is  put  for 
two  to  ten  minutes  into  a  stove   heated  to  not   more    than 
55°  to   60°  C.      The  sections  are  then  rinsed  several  times, 
with  water,  a  solution  of  one  part  of  toluidin  blue  in  3000 
of  water  is  poured  on   to  them,  they  are  again  stoved  for 
ten  minutes,  rinsed   with   water,  treated  with  96   per  cent, 
alcohol  till  no  more  colour  comes  away  (three   quarters  to 
two  minutes) ,  and  passed  through  absolute  alcohol  and  xylol 
into  xylol  balsam. 

For  the  very  elaborate  minutiae  described  by  the  author 
the  reader  must  consult  the  original. 


INDEX. 


The  numbers  refer  to  the  Pages. 


A. 


ABBE,  mounting  medium,  281. 

Absolute  alcohol,  71,  72,  81. 

Acanthocephali,  474. 

Acetate  of  copper,  65. 

Acetate  of  lead,  382. 

Acetate  of  potash,  for  bluing  haema- 
tein  stains,  181. 

Acetate  of  potash,  for  mounting,  271. 

Acetate  of  potash,  refractive  index,  81. 

Acetate  of  uranium,  36,  65. 

Acetic  acid,  action  in  fixing  mixtures, 
23—25,  49,  51,  62,  63;  fixing 
with,  62  ;  Lo  BIANCO'S  "  concen- 
trated," 62;  due  proportions  in 
mixtures,  63;  various  mixtures, 
63  et  seq. 

Acetic  acid  carmine,  Ifi9. 

Acetic  alcohol,  63,  382;  ditto  with 
sublimate,  64. 

Acetic  bichromate,  50. 

Aceto-carmine,  169. 

Acetone,  for  celloidin  imbedding,  123 

Acetone,  for  dehydration,  5. 

Acetone,  sublimate  solution,  57. 

Acid  alcohol,  72,  73. 

Acid  differentiation,  198,  203. 

"  Acid  "  dyes,  193. 

Acid  extraction,  198,  203. 

Acid,  free,  test  for,  see  Congo  red,  219, 
220. 

Acid  f  uchsin,  215. 

Acid  haemalum,  183. 

Acid  magenta,  215. 

Acid  rubin,  215. 

Acidophilous  mixture,  223. 


Acids,  see  Acetic,  Chromic,  Hydro- 
chloric, Nitric,  Osmic,  etc. 

Acids,  Congo  red  as  a  test  for,  219,  220. 

Actinida,  484. 

ADAMKIBWICS,  myelin  stain,  408. 

Adenoid  tissue,  437. 

Adipose  tissue,  437. 

Adjective  staining,  159. 

After-gilding,  251,  256. 

AGABABOW,  elastic  tissue,  441. 

AOASSIZ  and  WHITMAN,  pelagic  ova, 
346. 

Albumen,  examination  media,  271, 272. 

Albumen,  freezing  method,  139. 

Albumen,  injection  mass,  307. 

Albumen,  section-fixing  process,  143 — 
145,  147. 

Alcohol,  for  dehydration,  4 ;  for  pre- 
servation, 5,  272;  for  narcotisa- 
tion, 14 ;  for  fixing,  69—72  ;  for 
hardening,  70 ;  for  maceration, 
313  ;  removal  of,  6 ;  absolute,  71  ; 
methylated,  497;  acetic,  63,  64; 
one-third,  71 ;  hydrochloric  acid, 
72 ;  tests  for  purity,  70,  72 ;  table 
for  diluting,  498 ;  the  usual 
grades,  497. 

Alcoholic  carmines,  172 — 176. 

Alcoholic  cochineal,  174 — 176. 

Alcyonaria,  485. 

Aldehyde,  421. 

ALEXANDER,  reconstruction,  332. 

ALFEROW,  silver  impregnation,  246. 

ALFIERI,  bleaching,  328. 

Alizarin,  artificial,  227. 

ALLEN,  methylen  blue,  233. 


504 


INDEX. 


The  numbers  refer  to  the  Pages. 


ALT,  axis-cylinder  stain,  411. 
ALTMANN,  osnaicated  fat,   36;    osinic 

and  bichromic  mixture,  44 ;  nitric 

acid,   46;    paraffin    stove,     101; 

corrosion,  322 ;  fixatives  for  nuclei, 

363. 

Alum,  ammonia,  solubility,  184. 
Alum,  for  fixing,  53. 
Alum-carmine,  168,  169;    ditto  with 

picric  acid,  169. 
Alum-hffimatoxylin  stains,  180 — 187  ; 

general   characters,    180;  bluing 

them,  180. 
Amber  varnish,  292. 
AMBEONN  and  HELD,  polarisation,  406. 
Ammonia-alum,  184. 
Ammonia-carmine,  171. 
Ammonia-carmine,  how  to  neutralise, 

301. 

Ammonio-nitrate  of  silver,  246. 
Ammonium  molybdate  impregnation, 

261. 

Amphibia,  embryology,  341 — 344. 
Amphibia,  larvae,  357. 
Amphioxus,  346. 
Amphipoda,  embryology,  354. 
Amyl  alcohol,  for  clearing,  85. 
Amyl  nitrite,  297. 
Amyloid  matter,  210. 
AKDEEB,  phloroglucin,  325. 
ANDEES,    nicotin    narcotisation,    13; 

imbedding  squares,  93  ;  Actinida, 

484. 

ANDEEWS,  imbedding  apparatus,  93. 
Anilin,  for  clearing,  85. 
Anilin,  for  imbedding,  85. 
Anilin,  for  staining,  203. 
Aniliu  black,  226,  409. 
Anilin  blue,  224. 
Anilin  blue-black,  225,  409. 
Anilin  dyes,  generalities,  191  et  seq,  • 

and  see  Coal-tar  colours. 
Anilin  green,  223. 
Anilin  oil,  see  Anilin. 
Anilin  red,  207. 
Anilin,  refractive  index,  81. 
Anilin  water,  203. 
Aniseed  oil  freezing  mass,  139. 
Annelids,  470—472. 
Annelids,  embryology,  354. 


Anodonta,  16. 

Anthozoa,  484,  485. 

APATHY,  principles  of  technique,  10; 
washing  sublimate  material,  55 ; 
alcoholic  sublimate,  57;  osmic 
sublimate,  257 ;  paraffin  imbed- 
ding, 100;  knife  position,  105, 
106;  knife-holder,  107;  section- 
cutting,  112;  paraffin  mass,  116; 
gelatin  imbedding,  118 ;  celloidin 
imbedding,  121,  123,  124,  127, 
128 ;  serial  sections,  water  method, 
142 ;  methods  for  celloidin  sec- 
tions, 148,  149 ;  hffimatein  mix- 
ture I  A,  186 ;  alcoholic  hseina- 
toxylin  stain,  188;  methylen  blue, 
228,  231,  233,  234,  236,  237; 
cement  for  glycerin  mounts,  292  ; 
maceration,  318;  muscle  of  Ver- 
ines,  375 ;  gum  syrup,  236,  275 ; 
gold  chloride,  solutions,  243,  250  ; 
gold,  fore-gilding,  255;  gold, 
after-gilding,  257 ;  neuro-fibrils, 
394. 

A  PEL,  Gephyrea,  473. 

Araneida,  embryology,  352. 

Arctiscoida,  353,  469. 

Areolar  tissue,  435. 

ABNOLD,  neutral  red,  220  ;  blood,  449. 

AENSTEIN,  methylen  blue,  232,  235, 
236. 

ARONSON,  nerve  stain,  404;  ditto  with 
gallein,  408. 

Arsenic  acid,  325. 

Artefacts  of  fixation,  22,  52,  56. 

Arthropoda,  465 — 469. 

Arthropoda,  embryology,  349 — 354. 

Artificial  alizarin,  227. 

Artificial  fecundation,  330. 

Artificial  iodised  serum,  271. 

Artificial  saliva,  315. 

Ascaris,  ova,  64,  356. 

Ascidians,  459,  460. 

Ascidians,  gemmation,  346. 

Ascidians,  test-cells,  346. 

Asphalt  varnish,  290. 

Asphyxiation,  17. 

Astacus,  embryology,  353. 

Asteracanthion,  14. 

Asteroidea,  481. 


INDEX. 


505 


The  numbers  refer  to  the  Pages. 


Asteroidea,  larvae,  483. 
AUBEBT,  cements,  288. 
AUBURTIN,  serial  sections,  148. 
AUEBBACH,  staining  nerve-cells,  393  ; 

axis-cylinder  stains,  393,  411. 
AUGSTEIN,  Strongylus,  475. 
Aurantia,  223. 
Auricularia,  483. 
Aves,  embryology,  337 — 340. 
Axis-cylinder,  stains  for,  407—427. 
Axis-cylinder,  structure,  393. 
Azoschwarz,  226. 
AZOTJLAY,  osmic  acid  nerve  stain,  405; 

Golgi's  impregnation,  418. 

B. 

BABES,  safranin,  203,  204. 

BAKER,  C.,  address,  88. 

BALBIANI,  methyl  green    and    eosin, 

221  ;  embryological  methods,  331 ; 

Protozoa,  491,  494. 
BALBIANI    et    HENNEGUY,    Protozoa, 

491. 
BALLOWITZ,  muscle   of  Cephalopoda, 

374;  electric  organs,  434. 
Balsam,  Canada,  81,  137,  283,  284. 
Balsam,  Tolu,  81. 
BARFF'S  boroglyceride,  277. 
BARI,  Golgi's  impregnation,  420. 
Barium  bichromate,  49. 
BARNES,  Trichinae,  476. 
BABBOJS,  larvae  of  Echinouerms,  482. 
'•  Basic  "  dyes,  193. 
BASTIAN,  gold  method,  254. 
BATAILLON  and   KOEHLER,  methylen 

blue,  364. 
BAFMGABTEN,  bleu    de    Lyon,    225 ; 

f  uchsin  and  methylen  blue,  226 ; 

carmine  and  bleu  de  Lyon,  265. 
BAYEBL,  decalcification,  324;  cartilage, 

446. 
BEALE,   glycerin   jelly,   278;    shellac 

varnish,    293;     injections,    308; 

digestion,  319. 
BECK,  A.,  microtome,  104. 
HECK,  J.,  cements,  288. 
BECKER,  microtome,  88. 
BEDOT,  Siphonophora,  487. 
BEER,  medullated  nerve,  394. 


BEHRENS,  G.,  embryology  of  Sal- 
monidse,  345. 

BEHBENS,  W.,  Tabellen,  80;  refractive 
indices,  80 ;  cements,  288. 

BELA  HALLER,  maceration,  317. 

BELLABMINOW,  injection,  310. 

BELL'S  cement,  290. 

BENDA,  nitric  acid  fixation,  46 ;  iron 
hsematoxylin,  188  ;  copper  hseraa- 
toxylin,  191;  Lichtgrvin  stain, 
222;  Saureviolett  stain,  222. 

BENECKE,  stain  for  fibrils,  436. 

BENEDECENTI,  formol,  75. 

BENEDEN,  VAN,  sublimate  solution, 
55 ;  acetic  alcohol,  63  ;  mala- 
chite green,  222;  embryology  of 
rabbit,  335,  336;  embryology  of 
Tcenia,  355. 

BENEDIKX  and  KNECHT,  tar-colours, 
159. 

Bengal  rose,  221. 

Benzin  colophonium,  286. 

Benzoazurin,  207,  226. 

Benzol,  for  clearing,  85 ;  for  im- 
bedding, 96. 

Benzopurpurin,  220. 

Benzoyl  green,  222. 

Bergainot  oil,  for  clearing,  81,  83 ;  for 
imbedding,  97. 

BEBGONZINI,  plasma  cells  and  Mast- 
zellen,  439. 

BEBKLEY,  rapid  nerve  stain,  403 ; 
Golgi  impregnation,  419,  421. 

Berlin  blue  injections,  304,  309. 

BERLINERBLAU,  hsematoxylin,  398. 

BEBNABD,  maceration  of  mollusca,  465. 

BERNHEiM.gold  method,  255;  bladder 
of  frog,  376. 

BERNHEIMER,  retina,  432. 

BETHE,  treatment  of  osmic  material, 
34;  methyleii  blue,  237,  238; 
neuro-fibrils,  395,  502;  stain  for 
chitin,  469. 

BETTBNDOBF,  Distoma,  478. 

BETZ,  nervous  centres,  384. 

BEVAX  LEWIS,  see  LEWIS. 

BIANCO,  S.  Lo,  see  Lo  BIANCO. 

Bichloride  of  mercury,  tee  Sublimate. 

Bichromate  of  ammonia,  52,  383,  384, 
385. 


506 


INDEX. 


The  numbers  refer  to  the  Pages. 


Bichromate  of  barium,  49. 

Bichromate  of  potash,  generalities,  48 ; 
for  hardening,  49  ;  for  fixing  and 
hardening,  44,  50—52,  383;  for 
macei'ation,  316. 

Bichromate  of  silver  impregnation,  see 
GOLGI. 

Bichromate  and  osmic  mixtures,  44 ; 
other  mixtures,  50 — 52. 

Bichromate  material,  action  of  light 
on,  39. 

Bichromate  material,  bleaching,  50. 

BICKFALVI,  digestion,  319. 

Biebrich  scarlet,  221. 

BIEDEEMANN,  methyleii  blue,  233; 
nerve  and  muscle,  371,  372. 

BINET,  bleaching,  34 ;  ganglia  of 
Hexapods,  468. 

Biniodide  of  mercury  mounting  me- 
dium, 280. 

Bioblasts  of  ALTMANN,  365. 

BIONDI,  staining  mixture,  215 ;  blood, 
448. 

Bipinnuria,  483. 

Bismarck  brown,  infra  mi  am,  158. 

Bismarck  brown,  progressive,  210. 

Bismarck  brown,  regressive,  207. 

Bitume  de  Judee,  290. 

BIZZOZEEO,  gentian  violet,  205; 
blood-plates,  450,  452;  glands, 
453. 

BrzzozEEO  and  TOEEE,  blood,  450. 

BJELOTJSSOW,  injection,  307. 

Blackley  blue,  223. 

Bladder  of  frog,  375. 

BLANC,  Protozoa,  492. 

Blattida,  embryology,  352. 

Bleaching,  osmic  material,  34;  bi- 
chromate material,  50 ;  chromic 
material,  37,  38 ;  in  general,  327 
—329. 

Bleu  carmin,  226. 

Bleu  de  Lyon,  224. 

Bleu  de  nuit,  224. 

Bleu  lumiere,  224. 

BLOCHMAN,  serial  sections,  146;  ova 
of  Amphibia,  341 ;  Cestodes,  426, 
478;  Brachiopoda,  461. 

Blood,  447—453. 

Blue-black,  226. 


BLUM,  formol,  74,  75,  77;    celloidin. 

imbedding,  127. 

BOBEETZKT,  ova  of  Lepidoptera,  351. 
BOCCAEDI,   gold  method,  255;    stain 

for  nerve-cells,  391. 
BonM,  gold  method,  255. 
BOHM  and  OPPEL,  artificial  serum,  271 ; 

egg  of  fowl,  340 ;  nerve  stain,  404 ; 

Golgi  ditto,  419. 
BOHMEE,  honnatoxylin,  184. 
BOHMIG,  Turbellaria,  479. 
BOLTON,      haematoxyhn,     192,     402; 

Grolgi's  impregnation,  420. 
Bone,  442—445. 

BONNET,  embryology  of  dog,  337. 
Borax  carmine,  172. 
Borax  methylen  blue,  364. 
Bordeaux  R.,  219. 
BOEQEET,  paraffin  imbedding,  94. 
BOEN,    section-stretcher,    109;    recon- 
struction, 332 ;  orientation,  501. 
Boroglyceride,  277. 
BOTTIN,  picric  formol,  76. 
BOVEEI,   picro-acetic   acid,  67,   356; 

imbedding    small    objects,    332 ; 

medullated  nerve,  395. 
Brachiopoda,  461. 
BEADY,  chloral  hydrate,  272. 
BEAEM,  statoblasts,  317. 
Brain,  see  Neurological  methods. 
BEANDT,  glycerin  jelly,  278 ;  Infusoria, 

492 ;  Spluei-osoa,  494. 
BEASS,  paraffin,  116  ;  Protozoa,  493. 
BEAUEE,  embryology  of  scorpions,  353. 
BEAUN,  mounting  medium  for  Neimi- 

todes,     475;     Turbellaria,    479; 

Zoantharia  and  Alcyonaria,  485. 
BEAUS,  fixation  by   injection,  26;  ova 

of   Triton,  342  ;  liver,  457. 
Brazilin,  263. 
BEE<HJA,  logwood,  398 ;  nerve  stain, 

404. 

BEEMEE,  methylen  blue  and  eosin,  222. 
BEISTOL,  regeneration  of  osmic  acid, 

32 

BEOCK,  maceration,  316. 
BEODIE    and   KUSSELL,   blood-plates, 

452. 

Brown  cement,  290. 
BEOWN,  flagella,  496. 


INDEX. 


507 


The  numbers  refer  to  the  Pages. 

BBUCKE,  Berlin  blue,  304 ;  digestion,   j  CANFIELD,  iris,  375. 

319.  I  Caoutchouc  cement,  290. 

BROIL,  embryology  of  Diptera,  351.      j  Capitellidae,  14,  355. 


BBUK,  glucose  medium,  275. 
HEUXOTTI,  gelatin  imbedding,  118. 
Brunswick  black,  290. 
Bryozoa,  460  ;  statoblasts,  347. 
BUDGB,  injections,  310. 
BFEHLEB,  staining  nerve-cells,  393. 
BFMPUS,  celloidin  sections,  132. 
BFNGE,  stain  for  flagella,  495. 
BFRCI,  elastic  tissue,  441. 
BURCKHARDT,    E.,    bichromates,    49, 

382;  fixing   mixtures,   49;  pyro- 

ligneons-acid-carmine,  170 ;  pyro- 

ligneous-acid-haematoxyliu,    187 ; 

methyl    green,    209;     neuroglia, 

430. 
BFKCKHARDT,  R.,  brain  of  Prolopterus, 

387. 

BUBGER,  Xeuaertiua,  477. 
BFSCH,  osmic  mixture,  36,405;  decal- 

cification,  322,  323. 
BFSSE,  celloidin,  122,  124,  127. 
BiJTSCHLi,    paraffin    imbedding,    99; 

acid  haamatoxylin,  185  ;  iron  ha> 

matoxyliu,  191. 


C. 

CAJAL,  RAMON  Y.picro-indigo-carmine, 
265 ;  nerve-endings  in  muscle, 
373 ;  stain  for  nerve-cells,  391 ; 
Golgi  impregnation,  418;  meth\ - 
len  blue,  430;  retina,  432;  con- 
nective tissue,  435,  436. 

Cajeput  oil,  84. 

CALBERLA,  methyl  green,  208;  ditto 
and  eosin,  221 ;  indulin,  223 ; 
glycerin  mixture,  277;  artificial 
saliva,  315. 

Calcium  chloride  mounting  medium, 
271. 

CAIDWELL,  serial  sections,  146. 

CALLEJA,  picro-indigo-carmine,  265. 

Cambridge  rocking  microtome,  88. 

Canada  balsam,  index,  81 ;  for  im- 
bedding, 137;  for  mounting,  283, 
284;  as  a  cement,  292. 


CABAZZI,  peroxide  of  sodium,  34,  328; 
sublimate  liquid,  58;  ova  of 
Aplysia,  349 ;  Larnellibranchs, 
461. 

Carbolic  acid,  index,  81 ;  for  clearing, 
84. 

Carbolic  fuchsin,  207. 

Carbonic  acid  for  narcotisation,  17. 

Carmalum,  167 ;  with  indigo-carmine, 
264. 

Carmine,  generalities,  163  ;  analysis,. 
163;  stains  in  general,  166;  for- 
mulae for  stains,  167 — 176  ; 
aqueous,  167 — 172 ;  alcoholic,  172. 
— 176;  combination  stains,  264, 
265. 

Carmine  blue,  226. 

Carmine  solutions,  to  neutralise,  301. 

Carminic  acid,  163,  164,  166. 

CARNOY,  chromo-aceto-osmic  acid,  43  ; 
acetic  alcohol,  63,  64;  haemateiu 
stains,  181 ;  Congo  red,  220 ;. 
salt  solution,  269;  tannin  solution, 
273;  cement,  2y3;  micro-chemi- 
cal reactions,  359. 

CABNOY  and  LEBBUN,  ova  of  Am- 
phibia, 342;  ditto  of  Ascari*, 
356;  micro-chemistry,  360. 

CABPENTEB,  section  grinding,  13(3 ; 
cements  and  varnishes,  288. 

CABBIEEE,  eyes  of  Gastropoda,  462. 

CARTER,  injection,  303. 

Cartilage,  445,  446. 

CASTELLABNAF,  DB,  Ophiuridea,  482 ; 
Zoantharia,  484. 

CASTLE,  ova  of  Ciona,  346. 

Castor  oil  for  mounting,  287. 

CATTANEO,  palladium  chloride,  61  i. 
Protozoa,  493. 

Candina,  16. 

CAULLEBY,  Ascidians,  460. 

CAUSABD,  injection  of  spiders,  468. 

Caustic  soda  or  potash,  see  Soda  and 
Potash. 

CAVAZZAM,  hsematoxylin  and  Saure- 
fuchsin,  267. 

Cedar-wood  oil,  index,  81 ;  for  clearing,. 


508 


INDEX. 


The  numbers  refer  to  the  Pages. 


81 ;  imbedding,  97 ;  for  pre- 
serving, 6  ;  for  mounting,  286  ; 
for  dissecting  in,  9. 

Celloidin  imbedding,  120 — 135  ;  gene- 
ralities, 120,  121 ;  preliminary 
preparation,  121 ;  celloidin  bath, 
122;  imbedding,  123;  orienta- 
tion, 124;  hardening,  125—128; 
preserving  blocks,  128 ;  fixing 
blocks  to  microtome,  128;  cutting, 
128 ;  clearing  and  mounting,  131 ; 
resume,  131 ;  the  new  method, 
132 ;  Gilson's  rapid,  132 ;  the  dry 
cutting  method,  133 ;  celloidin 
and  paraffin  method,  134. 

Cells,  paper  for  mounting,  289. 

Cements  and  varnishes,  288  et  seq. 

Central  corpuscles,  centrosomes,  etc., 
364. 

Central  nervous  system,  see  Nervous 
centres  and  Neurological  methods. 

Cephalopoda,  embryology,  347 ;  eyes, 
463. 

Cercarise,  478. 

Cerebrum,  cerebellum,  see  Neurological 
methods. 

CEBFONTAINE,  Lumbricus,  470. 

CEETES,  Infusoria,  491—493. 

Cestodes,  477  ;  embryology,  355. 

Chaetopoda,  470-472. 

CHEATLE,  dehydration  apparatus,  4. 

Chemicals,  161,  162. 

Chemistry  of  tar-colours,  159. 

CHENZINSKY,  methylen  blue  and  eosin, 
222 

CHICHKOFF,  Turbellaria,  479. 

Chilopoda,  466. 

Chinablau,  225. 

China  blue,  225. 

Chinolinblau,  chinolin  blue,  223. 

Chironomus,  ova,  351. 

Chitin,  466,  469. 

Chiton,  embryology,  349. 

Chloral  hydrate,  for  narcotisation,  14, 
15;  preservative  solutions,  272, 
274,  279;  for  preserving  injec- 
tions, 298;  for  maceration,  314. 

Chlorate  of  potash  for  maceration,  317. 

Chloride  of  aluminium  carmine,  167. 

Chloride  of  calciuinfor  mounting,  271. 


j   Chloride  of  copper  fixative,  65. 

I   Chloride  of  gold,  see  Gold  chloride. 

!   Chloride  of  iridium,  61. 

I   Chloride  of  magnesium,  narcotisation, 

16. 

!   Chloride  of  manganese,  269. 
I   Chloride  of  palladium,  for  fixing,  61 ; 

for  staining,  260,  407. 
!   Chloride  of  platinum,  60;    mixtures, 

44,  45,  48,  331. 
|   Chloride  of  sodium,  see  Salt. 
i   Chloride  of  vanadium  staiu,  410. 

Chloride  of  zinc,  for  fixing,  61,  383. 
I  Chlorine  for  bleaching,  327. 
Chloroform,  for  narcotisation,  13  ;  for 
clearing,  85;   for  imbedding,  97, 
99 ;  for  hardening  collodion,  126. 
i  Choroid,  328. 

i   Chromate,  neutral,  of  ammonia,  52. 
I   Chromate  of  lead  impregnation,  260. 
Chromate  of  silver,  see  GOLGI. 
Cnromates  for  fixing  and  hardening, 

48. 

Chromatin,  reactions,  359. 
Chromatiu   stains,   defined,  153 ;    the 
coal-tar,   202—211 ;    cytological, 
363. 

Chromic  acid,  generalities,  36 ;  fixing 
with,     37;      washing     out,     37; 
hardening    with,    38;    action   of 
light,  39;    mixtures,  39  et   seq., 
46   et  seq.,  49,   59,  60,  69;    for 
maceration,  316;   for  decalcifica- 
tion,  323—325. 
Chromo-acetic  acid,  40. 
Cliromo-aceto-osmic  acid,  40 — 44;  for 

decalcification,  325. 
Chromo-formic  acid,  40. 
Chromo-nitric  acid,  46;  for  bleaching, 

329. 

Chromo-osmic  acid,  40. 
Chromo-platinic  mixture,  48. 
Chromo-sublimate,  59. 
CHRSCHTSCHONOWIC,     gold     method, 

254. 
CIACCIO,  gold  method,  254 ;  corpuscles 

of  Golgi,  374. 

CIAGLINSKI,  myelin  stain,  408. 
Cilia  of  Infusoria,  495. 
Ciliated  epithelium,  464. 


INDEX. 


509 


The  numbers  refer  to  the  Pages. 
Cinnamon  oil,  81,  83.  '   Congo  red,  219. 


CLABKE'S  spirit-proof  cement,  290. 
Clasnmtocytes,  440. 
CLATPOLE,  Mayer's  albumen,  144. 
Cleaning  slides  and  covers,  499. 
Clearing,  generalities,  6,  78  et   seq. ; 
clearing  agents,  80  et  seq. ;  practice 
of,  79;    choice   of  an  agent,  81, 
130;  celloidin  sections,  130. 
Clove  oil,  for  minute  dissections,  9,  82  ; 
refractive  index,  81;  for  clearing, 
82;     for     imbedding,     96;     for 
differentiating  stains,  199. 
Coal-tar  colours,  193  et  seq.  ;  regres- 
sive staining  with,  196;  progres- 
sive  ditto,  208 ;    choice  of,  201  ; 
chromatin      stains,       202—211  ; 
plasma  stains,  212—227. 
COBB,   differentiator,   4 ;    Nematodes, 

475. 

Cocaine,  narcotisation,  15. 
Coccida?,  467. 

Cochineal,  generalities,  165 ;  aqueous, 
PABTSCH'S.  168;  CZOKOB'S,  168; 
RABL'S,  169  ;  alcoholic,  MAYEB'S, 
174—176. 
Cochlea,  433,  434. 
COE,  Miracidia,  356. 
Ccelenterata,  484—489. 
Ccerulein,  223. 

COHNHEIM,  gold  method,  251. 
COLE,  freezing  method,  138. 
Coleoptera,  embryology,  352. 
COLES,  blood,  447. 
Collagen,  436. 
COLLIN,  Criodrilus,  470. 
COLLIKOE,  pelagic  ova,  346. 
Collodion  for  fixing  sections,  145,  146, 

149. 
Collodion    imbedding,   120 — 135,    and 

see  Celloidin. 

Collodionisation  of  sections,  110. 
Colophonium,  for  imbedding,  137  ;  for 

mounting,  285 ;  cement,  292. 
COLUCCI,  retina,  433. 
Combination  stains,  262  et  seq. ;  car- 
mine combinations,  264  ;  haema- 
tein  ditto,  266. 
Congelation  imbedding  methods,  137. 


CONKLIN,  ova  of  Crepidula,  349. 
Connective  tissues,  435  et  seq. 
CONSEB,  Rotatoria,  474. 
Copal  section  method,  136. 
Copepoda,  466;  embryology,  354. 
Copper,  sulphate,  51—53,  487;  chlo- 
ride and  acetate,  65. 
Copper  hseinatoxylin,  191. 
Coral,  484,  485. 
Corallin,  207. 

COEI,  narcotisation,  14 ;  cocaine,  15 ; 
keeping  osmic  acid,  32 ;  chromo- 
aceto-osmic  add,  41. 
Cornea,  243,  369. 
COBBING,    Krohnthal's  impregnation, 

428. 

Corpuscles,   tactile,  368;     of    Herbst 
and  Grandry,  368 ;    of  Meissner, 
252 ;  of  Golgi,  373 ;  of  Meissner 
and  Krause,  369. 
Corrosion,  321,  322. 
Corrosive  sublimate,  see  Sublimate. 
Coupler's  blue,  223. 
Cox,  Nissl's  stain,  391,  392;   medul- 
lated  nerve,  394;    sublimate  im- 
pregnation, 426. 

Creasote,  index,  81  ;  for  clearing,  85.. 
Crinoidea,  482. 
Cristatella,  347. 
Crocein,  214. 
Crystalline,  370. 
Ctenopbora,  489. 
CUCCATI,  retina,  432. 
Cupric    sulphate   for    fixing,   51 — 53V 

487  ;  for  staining,  260. 
Curarisation,  357. 

CUBSCHMANN,  amyloid  matter,  210. 
Cyanin,  223. 

CTBULSKY,  gold  impregnations,  258. 
Cyclops,  ova,  354. 

Cytological  methods,  357 — 365  j  living 
cells,  357  ;  fresh  cells,  358 ;  micro- 
chemical  reactions,  359 ;  fixing 
agents,  360 ;  chromatin  stains, 
363  ;  plasma  stains,  364,  nucleoli 
and  granules,  365. 

CZOKOB,  cochineal,  168;  turpentine- 
cement,  291. 


"510 


INDEX. 


The  numbers  refer  to  the  Pages. 


DADDI,  fat,  437. 

DAHLGREN,  double  imbedding,  135. 

Dahlia,  206. 

Damar  (dammar),  gum,  284. 

DAVIDOFF,    ova    o£    Distaplia,  346 ; 

Siphonophora,  488. 
DAVIES,  injection,  303. 
De-alcoholisation,  6,  78  et  seq. ;  choice 

of  an  agent,  81. 
DEANE'S  medium,  274;  glycerin  jelly, 

278. 

Decalcification,  322—326. 
DECKER,  section-stretcher,  109. 
DEECKE,  encephalon,  385. 
DE  FILIPPI,  Tcenia,  477. 
DE  GROOT,  serial  sections,  140. 
Dehydration,  3 — 5. 
DEKHTJYSEN,  silver  impregnation,  246 ; 

fat,  437. 

DELAFIELD,  hrcmatoxylin,  184. 
DELAGE,   Turbellaria,  479;   larvae   of 

sponges,  490. 

DELLA  VALLE,  ova  of  Orchestia,  354. 
Deltapurpurin,  220. 
DENDY,  Geonemertes,  477. 
Depigmentation,  466,  467. 
DE  QUERVAIN,  nervous  tissue,  379. 
Desilicification,  326. 
DE  SOUZA,  pyridin,  73. 
DETERMANN,  blood-plates,  452. 
DEWITZ,  injection  of  molluscs,  464. 
Dextrin  freezing  mass,  138. 
DEZSO,  sponges,  490. 
Differentiation,  optical,  by  fixation,  23, 

41. 
Differentiator,   Cobb's,   4;    Haswell's, 

4 ;  Cheatle's,  4. , 
Diffusion  apparatus,  4. 
Digestion,  319,  320,  360. 
DIMMER,  serial  sections,  152. 
DIMMOCK,  carminic  acid,  163. 
DIOMIDOFF,  nervous  tissue,  383. 
Diptera,  embryology,  351. 
Dissections,  minute,  6,  9. 
Dissociation,  methods  of,  312. 
Distaplia,  346. 
Distomum,  Miracidia,  356. 
DOGIEL,  methylen  blue,  231,  233,  235, 

236,     238,     239;     corpuscles     of 


Herbst  and  Grandry,  368 ;  cor- 
puscles of  Krause,  369  ;  iris,  375  ; 
retina,  432. 

DOLLKEN,  formol  imbedding  method, 
139;  soap  imbedding,  117;  nerve 
stain,  401. 

DONALDSON,  nervous  centres,  383. 

DOSTOIEWSKY,  iris,  375. 

Double  imbedding,  134. 

Double  stains,  see  Stains,  combined. 

DOYERE,  Arctiscoida,  469. 

DRASCH,  gold  impregnation,  251. 

"  Drittelalcohol/'  71. 

DHOST,  epithelium  of  mollusca,  465. 

DBUEBIN,  blood-plates,  452. 

DRUENER,  fixation  by  injection,  26; 
osmic  sublimate,  59. 

DUBOSCQ,  Chilopoda,  466 ;  blood,  449. 

DUNHAM,  celloidin  sections,  131. 

Du  PLESSIS,  Nemertians,  476;  Pro- 
tozoa, 493. 

DTJRIG,  Golgi's  impregnation,  419. 

DTTVAL,  section-flattening,  113 ;  collo- 
dion imbedding,  121 ;  silver  im- 
pregnation, 245,  248;  carmine 
and  anilin  blue,  265;  embryology 
of  birds,  337,  339. 

E. 

Ear,  inner,  433 

Eau  de  Javelle,  321,  328,  341 ;  Eau  de 
Labarraque,  321,  328. 

EBNER,  VON,  decalcification,  324. 

Echinodermata,  480—484. 

Echtgelb,  214 

Echtgrun,  394 

EDINGER,  liquid  of  Erlicki,  52 

Eggs,  of  fowl,  337;  and  see  Embry- 
ological  methods. 

EHLERS,  fixative,  40 

EHRENBATJM,  section  grinding,  137. 

EHRLICH,  acid  hsematoxylin,  185 ;  clas- 
sification of  tar- colours,  193  ; 
stain  for  bacteria,  205;  triacid 
mixture,  218;  acidophilous  mix- 
ture, 223 ;  mixture  C  (eosino- 
philous),  223  ;  indulin-aurantia- 
eosin,  223 ;  neutral  red,  220  ; 
methylen  blue,  229 ;  Mastzellen , 
437;  leucocytes,  451. 


INDEX. 


511 


The  numbers  refer  to  the  Pages. 


"EHRLICH-BIOXDI-HEIDENHAIN  stain, 
215. 

EHRMANN  and  JADASSOHN,  plasma 
fibrils,  367. 

EICHLEK,  labyrinth,  434. 

EISEN,  iridium  chloride,  61;  ruthe- 
nium red,  261;  Braziliu,  263; 
gum  Thus,  287. 

EisiG,  alcohol  narcotisation,  14;  Capi- 
tellidae,  14,  471 ;  chrorao-platinic 
mixture,  48;  maceration,  316; 
embryology  of  Capitella,  355. 

EISMOND,  quieting  Infusoria,  491. 

EKMAN,  Brachiopoda,  461 

Elastic  tissue,  440 ;  of  spleen,  457. 

Electric  organ?,  434. 

Electrification  of  paraffin,  113. 

ELSCHNIG,  celloidin,  122. 

Embryological  methods,  330—356; 
generalities,  330 — 333;  Amphibia, 
341—344 .  Arthropoda,  349— 
354;  Aves,  337—340;  Bryozoa, 
347  ;  Echinodermata,  482  ;  Mam- 
malia, 333—337;  Mollusca,  347 
—349;  Pisces,  344—346;  Rep- 
tilia,  340 ;  Tunicata,  346  ;  Vermes, 
354—356. 

KMERY,  injection,  310. 

Encephalon,  see  Neurological  methods. 

Endosmosis,  4. 

ENGELMANN,  epithelium,  464. 

Enteropneusta,  469. 

Entire  objects,  preparation  of,  8. 

E.NTZ,  GEZA,  Protozoa,  492. 

Eosin,  221 ;  \\  ith  haematoxylin,  266. 

Eosinophilous  mixture,  223 

Epidermis,  366. 

Epithelium,  366 ;  renal,  458  ;  ciliated, 
464. 

ERLANGEB,  TON,  Tardigrada,  353  ;  ova 
of  Ascaris,  356. 

ERLICKI,  liquid  of,  51. 

ERMENGEM,  VAN,  stain  for  flagella,496. 

ERREBA,  uigrosin,  207. 

Erythrosin,  221. 

Essences,  see  Oil,  and  Clearing. 

ETEBNOD,  paraffin  blocks,  108  ;  histo- 
logical  rings,  244. 

Ether,  for  narcotisation,  14,  357  ; 
for  preserving,  5. 


EVERARD,    DEMOOR,    and    M  ASSART, 
haematoxylin  and  eosin,  266. 

EWALD,  capillary  siphon,  4 ;   section- 
washing  apparatus,  4  ;  blood,  449. 

EWING,  Nissl's  stain,  391 
•  Examination  and  preservation  media, 
watery,  268  —  272 ;  mercurial, 
272,  273;  various,  273—276; 
glycerin,  276—278;  jellies,  278 
—280;  resinous,  281— 287;  high 
refractive  liquids,  280.  281. 

EXNER,  medullated  nerve-fibres,  404. 

ETCLESHTMER,    celloidin    imbedding, 
124, 132. 

Eyes,  of  Mollusca,  462, 463  ;  of  Arthro- 
pods, 467 ;  of  Asteroidea,  481. 


F. 

FABBE-DOMEBGUE,  glucose  medium, 
275 ;  Protozoa,  492,  494. 

FAIBCHILD,  washing  cylinders,  4. 

FABIS,  glycero-gum,  274. 

FABBANTS,  mounting  medium,  274. 

Fat,  437. 

Fatty  bodies,  blackening  by  osmic 
acid,  36,  437. 

Fecundation,  artificial,  330. 

FEIST,  methylen  blue,  237;  spinal 
cord,  389. 

FELIX,  embryology  of  Salmonidae,  345. 

FERRERI,  decalcification,  325,  326. 

Ferricyanide  of  potassium  for  bleach- 
ing, 35. 

FERRIES,  blood,  451. 

Fibrin,  stains  for,  452. 

KICK,  Golgi's  impregnation,  422. 

FIEDLER,  Spongilla,  489. 

FIELD  and  MARTIN,  paraffin  imbed- 
ding, 97,  104;  double  imbedding, 
134;  serial  sections,  145. 

FILIPPI,  DE,  Tamil,  477. 

FINOTTI,  Marchi's  nerve  st«in,  405; 
myeliii  stain,  408;  axis-cylinder 
stain,  411. 

FISCHBL,  medullated  nerve,  395. 

FiSCHEB,  A.,  Fixirtinff,  Farbuiy,  und 
Sau  des  Protoplasmas,  21 ;  theory 
of  fixation,  22—25 ;  ditto  of  stain- 
ing, 158;  haomatein  stains,  180; 


512 


INDEX. 


The  numbers  refer  to  the  Pages. 


methyl    green,    209;    leucocytes, 
452.  " 

FISCHER,  E.,  gold  method,  252 ;  tac- 
tile corpuscles,  368 ;  nerve  and 
muscle,  372. 

FISCHER,  P.  M.,  soap-imbedding,  for 
Trematodes,  478. 

FISH,  oil  of  thyme,  84  ;  celloidin  im- 
bedding, 123 ;  decalcification,  322, 
324 ;  hardening  nervous  tissue, 
383,  385,  386,  388;  Golgi's  im- 
pregnation, 420. 

Fixation,  2,  19,  26,  360  et  seq. ;  by  in- 
jection, 378,  421 ;  of  marine 
animals,  27. 

Fixation  images,  21. 

Fixation  precipitates,  21,  361. 

Fixing  agents,  action  of,  21 ;  characters 
of  the  usual,  23 ;  choice  of,  for 
beginners,  25 ;  the  various,  31 — 
77;  cytological,  360. 

Flagella,  495. 

FLATAU,  hardening  brain,  383 ;  Golgi's 
sublimate  method,  426. 

FLECHSIG,  gold  method,  255;  nerve 
stain,  404  ;  Golgi's  impregnation, 
427. 

FLEMMING,  preservation,  5;  chromo- 
acetic  acid,  40 ;  chromo-aceto- 
osmic  acid,  40 — 44 ;  action  of  bi- 
chromate, 48 ;  picro-osmic  acid, 
69;  saf ranin,  203 ;  gentian,  204; 
dahlia,  206;  picric  acid,  212; 
Orange  G,  213;  Orange  method, 
213,  214;  damar  solution,  284; 
epithelium,  366;  fat,  437;  bone, 
445;  goblet-cells,  455;  eyes  of 
Gastropoda,  462;  injection  of 
Acephala,  464. 

FLESCH,  chromo-osmic  acid,  40 ;  Wei- 
gert's  nerve  stain,  398 ;  inner  ear, 
434. 

FLORMAN,  celloidin,  127. 

Fluorides,  61. 
FOA,  fixing  mixture,  60. 
FOETTINGER,  narcotisation,  14. 
FOL,  narcotisation,  17;  treatment  of 
osmic  material,  34,  35 ;  chromo- 
aceto-osmic  acid,  41 ;  nitric  acid, 
46 ;      perchloride    of    iron,    61  ; 


picro-chromic  acid,  69  ;  vacuum 
imbedding,  101 ;  serial  sections, 
146 ;  gold  impregnation,  258 ; 
iron  stain,  260;  glycerin  jellies, 
279  ;  injections,  metagelatin,  298  ; 
injections,  carmine,  302;  injec- 
tions, Berlin  blue,  305  ;  injections, 
brown  and  yellow,  306  ;  decalcifi- 
catiou,  325 ;  reconstruction  of 
sections,  332 ;  Tintinnodea,  493. 
Fore-gilding,  251. 

Formaldehyde,  73 ;  as  a  mordant,  200 ; 
for  maceration,  314 ;  for  nervous 
tissue,  385 — 387;  for  the  Golgi 
impregnation,  419,  420;  and  see 
Formol. 

Formalin,  see  Formaldehyde. 
Formalose,  see  Formaldehyde. 
Formic  acid,  65. 
Formic  acid  carmine,  170. 
Formol,    generalities,    73 ;     for    pre- 
serving, 74;   for  hardening,    74, 
76,  77,  385—387  ;  for  fixing,  75  ; 
for  hardening  celloidin,  127  ;   as 
an  antiseptic,  77 ;    as  an  imbed- 
ding mass,  139 ;    as    a  reducing 
agent,  76,  77 ;  as  a  mordant,  200  ; 
for  gelatin  imbedding,   119;    for 
the  Golgi  impregnation,  419. 
"  Formol-Muller,"  76. 
Fowl,  embryology  of,  337—340. 
FRANCOTTE,  vacuum  imbedding,  101 ; 

section-stretcher,  109. 
FRANKL,  imbedding  box,  93. 
FREEBORN,  connective  tissue,  435. 
Freezing  section  method,  137 — 139. 
FRENKEL,  palladium   chloride   liquid, 

61. 

FRENZEL,  sublimate  solution,  57. 
FREY,   H.,   indifferent    liquids,   269 ; 
artificial  serum,  271 ;  white  injec- 
tion, 306. 

FRET,  M.,  peripheral  nerves,  406. 
FRIEDLAENDER,  Golgi's  impregnation, 
413  ;    cupric    sulphate    fixation, 
488. 

FRIEDMANN,  nerve  stain,  403. 
Fuchsin,  basic,  207 ;  acid,  215. 
Fuchsin  and  methylen  blue,  226. 
Fuchsin  S,  215. 


INDEX. 


513 


The  numbers  refer  to  tire  Pages. 


FircHS-WoLFRiXG,  glands  of  larynx, 

456. 
FUSARI,  cartilage,  446. 

G. 

GAD  and  HEYMANS,  polarisation,  406. 

GAGE,  picric  alcohol,  69 ;  clearing 
mixture,  85 ;  section-stretcher, 
109  ;  relloidin  sections,  132,  147  ; 
albumen  fluid,  272 ;  starch  injec- 
tion. 311;  maceration,  314,  316, 
317. 

GALEOTTI,  intra  vitam  staining,  157 ; 
neutral  red,  220. 

Gallein,  408. 

GALLEMAERTS,  serial  sections,  145. 

GALLI,  neuroceratin,  393. 

Gamboge  injection,  309. 

GARBINI,  safranin,  204 ;  Alcyonaria, 
485. 

GARDINER,  ova  of  Polychoerus,  355. 

GARDNER,  elastic  tissue,  441. 

GASKELL,  section-flattening,  113. 

Gastropoda,  462—464;  embryology, 
348. 

<iAULE,  fixing  liquid,  57;  serial  sec- 
tions, 140. 

GAT-LrssAC,  table  for  diluting  alco- 
hol, 498. 

GEBERG,  gold  method,  255  ;  corpuscles 
of  Herbst,  369. 

GEBHARDT,  crystalline,  370. 

GEDOELST,  digestion,  320 ;  medullated 
nerve,  394. 

GEHTTCHTEN,  VAX,  acetic  alcohol,  63; 
sectioning  nervous  system,  389  ; 
Xissl's  stain,  390;  medullated 
nerve,  393  j  Golgi's  impregnation, 
420. 

Gelatin,  imbedding,  117 — 119  ;  freez- 
ing mass,  138 ;  section  fixative, 
146;  injection  masses,  297—306; 
injection  masses,  carmine,  300 — 
303  ;  injection  masses,  blue,  303 
—305  ;  other  colours,  305,  306. 

Gelatin  cement,  289. 

Gemmation  of  Ascidians,  346. 

Gentian  violet,  204. 

GEOFFROY,  gelatin  medium,  280. 


Gephyrea,  472. 

GERATTLD,  narcotisation,  16 ;  Caudina, 

481. 

GERLACH,  J.,  carmine  injection,  303  ; 
gold  method  for  spinal  cord,  256. 
GERLACH,  L.,  glycerin  jelly,  118;  em- 
bryology  of    birds,   337  j    nerve- 
endings  in  muscle,  372. 
i  GEROTA,  formal,  75,  387;    silver  im- 
pregnation, 248 ;  VVeigert's  nerve 
stain,  398;  Golgi's  impregnation, 
420. 

GIACOMINI,  serial  sections,  152;  pre- 
serving brains,  430. 
GIERKE,  impregnation,  242 ;  haema- 
toxylin  and  eosin,  266;  macera- 
tion, 315  ;  anilin  blue-black, 
409. 

GIESBRECHT,  clearing,  79  ;  imbedding 
squares,  93;  paraffin   imbedding, 
99;  section  grinding,  137;  serial 
sections,  146 ;  Copepods,  466. 
GlESON,     VAN,     origanum     oil,     83  ; 
picro-Saurefuchsin,    215;    Saure- 
fuchsin  with   hsematoxylin,  267 ; 
formalin  for  nerve  tissue,  386. 
GiGLio-Tos,  blood,  447,  451. 
GiLSON,    mercuro-nitric   fixing    fluid, 
57;  zinc  chloride  fixing  fluid,  61; 
acetic  alcohol  with  sublimate,  64 ; 
bleaching     bichromate     material, 
50 ;    acetate    of     uranium,    65 ; 
rapid  celloidin  method,  132  ;  mer- 
curial examination   liquid,   272  ; 
glycerin  jelly,  279. 
Glands,  453—458. 
Glass,  refractive  index,  81. 
Glucose  mounting  media,  275. 
Glycerin,  refractive    index,   81,   276 ; 
mounting  media,  276—279 ;  extra 
refractive,  276 ;  method  of  mount- 
ing in,  276,  289. 

Glycerin  and  alcohol  mixtures,  277. 
Glycerin  injections,  299,  308,  309. 
I  Glycerin  jellies,  for   imbedding,  118; 

for  mounting,  278,  279. 
Glycero-gum,  274. 
Glychsemalum,  184. 
GOADBY'S  fluids,  273. 
Goblet-cells,  453,  455. 

33 


514 


INDEX. 


The  numbers  t 

GOETTE,  hardening  ova,  34-3. 

Gold  chloride,  impregnations,  249 ; 
commercial  salts,  250;  fore-gild- 
ing, 251—256 ;  after-gilding,  256 
— 258  ;  marine  animals,  258 ;  pre- 
serving preparations,  258;  and  see 
the  names  of  authors. 

Gold  orange,  214. 

Gold  size,  290. 

GOLDSCHEIDER  and  FLATAU,  Nissl's 
stain  391. 

GOLGI,  chromate  of  silver  impregna- 
tion :  introduction,  411 — 414 ; 
slow  process,  414 — 416 ;  rapid 
process,  416;  mixed  process,  416; 
critique  of  the  same,  417  ;  varia- 
tions, 418 — 424;  gold  method, 
256;  fixation  by  injection,  378; 
bichromate  and  sublimate  method, 
424. 

GOLGT,  corpuscles  of,  373,  374. 

GOODALL,  spinal  cord,  379. 

Goodsiria,  347. 

GORDON,  stain  for  flagella,  496. 

GORONOWITSCH,  embryology  of  Sal- 
monidae,  345. 

GOTHARD,  Nissl's  stain,  390. 

GRABERG,  stain,  219. 

GRAF,  formol,  76 ;  Hirudinea,  472. 

GRAFF,  VON,  Turbellaria,  479. 

GRAHAM,  Trichinae,  476. 

GRAM,  staining  method,  205. 

GRANDIS,  Mayer's  albumen,  145. 

GRANDRY,  corpuscles,  368. 

Granule  cells,  437 — 440 ;  and  see  Leu- 
cocytes. 

Granules,  365. 

Grape-sugar  imbeddiixg,  135. 

Graphic  or  plastic  reconstruction,  332  ; 
see  Orientation. 

GRASER,  staining  method,  207,  211. 

Gregarinae,  494. 

GRENACHER,  alum-carmine,  168;  borax- 
carmine,  172 ;  hydrochloric  acid 
carmine,  174;  haeniatoxylin,  184; 
castor  oil  for  mounting,  287; 
bleaching  mixture,  328 ;  eyes  of 
molluscs,  463. 

GREPPIN,  Golgi's  impregnation,  423. 

GRIEB,  alum-carmine,  168. 


efer  to  the  Pages. 

GRIESBACH,     metanil     yellow,     214 ; 

Sauregelb,    Echtgelb,    Tropaeoliii 

0,   crocein,    gold     orange,    214; 

Congo  red,  219;    benzopurpurin, 

220  ;  Biebricher    Scharlach,  221 ; 

Bengal    rose,   221 ;  iodine   green, 

222;    elastic  tissue,  440;    blood, 

449. 

GROOT,  DE,  serial  sections,  140. 
GROUVEN,  Ehrlich-Biondi  stain,  217. 
GRUBLER    and     HOLLBORN,     address, 

162  ;  anilin  blue-black,  226 ;  salts 

of  gold,  250. 
Griinpulver,  208. 
Grunstichblau,  224. 
GUDDEN,  Pal's  nerve  stain,  401. 
GUEGUEN,  methyl  salicylate,  86. 
GUIGNET,  injection,  305. 
GULLAND,      section-flattening,      113 ; 

serial    sections,  140,  151;    blood, 

447. 
Gum,  imbedding,  135;    for   freezing, 

138 ;    mounting   media,  274,  275 ; 

injection  mass,  307;  mucilage  for 

labels,  500. 
Gum  damar,  284. 

Gum  sandarac  for  mounting,  287. 
Gum,  Thus,  287. 
GUNTHER,  elastic  tissue,  441. 


H. 


HAECKER,  ova  of  Cyclops,  354 ;  micro- 
chemistry  of  the  cell,  360. 

Haeinacalcium,  185. 

Hamvalum,  182,  183. 

Haemalum  and  indigo-carmine,  264. 

Haemateate  of  ammonia,  180. 

Hannatein,  generalities,  177,  179,  182  ; 
formulae  for  stains,  182 — 192; 
and  see  Haematoxylin. 

Haematoxylin,  generalities,  177;  alco- 
holic stock  solution,  179 ;  UNNA'S 
solution,  179 ;  characters  of  alum- 
haematoxylin  stains,  180;  formulae 
for  stains,  182 — 192;  the  alum 
compounds,  182 — 187;  other  com- 
pounds, 187—192. 

Haematoxylin  and  eosin,  266. 


INDEX. 


515 


The  numbers  refer  to  the  Pages. 


Hseinatoxyliu  aud  picro-Saurefuchsin, 

267. 

Haematoxylin  and  safranin,  267. 

Haematoxyliu  and  Saurefuchsin,  267. 

Haematoxylin  Combination  stains,  266. 

HAENSEL,  liquid  of,  69. 

Hair,  368. 

HALLE  and  BOBN,  celloidin  imbedding, 
125. 

HALLEB,  BELA,  maceration,  317. 

HAMANN,  Acanthocephali,  474;  Aste- 
roidea,  481. 

HAMBURGER,  salt  solution,  269. 

HAMILTON,  freezing  method,  138; 
hardening  brain,  384. 

HANDWERCK,  osmicated  fat,  36. 

HANSEN,  hsematoxylin,  184;  connec- 
tive tissue,  435;  elastic  tissue, 
441. 

HANTSCH,  glycerin  liquid,  277. 

Hardening,  generalities,  28 ;  practice 
of,  29  ;  nerve  centres,  379—388. 

Hardening  agents,  see  Fixing  agents. 

HARDY,  Rotatoria,  473. 

HARMER,  silver  impregnation,  248. 

HARRIS,  haematoxylin,  184;  toluidiu 
blue,  208,  240;  methyleu  blue, 
239;  thionin,  240;  nerve  stain, 
404. 

HARRISON,  embryology  of  Salmonidae, 
345. 

HAKTI>*G,  calcium  chloride,  271 ;  white 
injection,  306;  gamboge  injection, 
309. 

HASSELAER,  object-holder,  89. 

HASWELL,  dehydration  apparatus,  4. 

HAUG,  decalcification,  322,  325,  326 ; 
nerve  stain,  404. 

HATEM,  blood,  447,  449. 

Heat,  for  killing,  11,  12. 

HEIDENHAIN,  M.,  sublimate  solution, 
57;  paraffin  imbedding,  97,  100, 
116 ;  serial  sections,  140 ;  iron- 
hsematoxylin,  189;  Ehrlich-Biondi 
stain,  216,  217;  Bordeaux  R, 
219 ;  vanadium  haematoxylin, 
365. 

HEIDENHAIN,  R.,  chrome  haematoxylin, 
187  ;  Ehrlich-Biondi  stain,  215. 

HEIDER,  paraffin  sections,  112. 


HELD,  sublimate  solution,  57;  stain 
for  nerve-cells,  391 ;  knife-warmer, 
501 ;  iron  heematoxylin,  502. 

HELLER  and  GUMPERTZ,  medullated 
nerve,  260,  406. 

HENCHMAN,  ova  of  Limax,  348. 

HENKING,  section  cutting,  111 ;  em- 
bryology of  Arthropoda,  350 — 
352;  examination  liquid,  351. 

HENNEGUY,  object-holder,  89 ;  section 
flattening,  113  ;  section-fixing* 
144;  alum-carmine,  169;  per- 
manganate method,  200;  embryo- 
logical  methods,  333—336,  344, 
348;  Protozoa,  491,  492;  re- 
staining  old  mounts,  500. 

HENOCQUE,  gold  method,  254. 

HERBST,  corpuscles,  368. 

HERMANN,  platino-aceto-osmic  acid, 
45  ;  formalin,  74—76 ;  safranin 
and  gentian  stain,  205;  osmic 
acid  and  pyroligneous  acid  stain, 
259 ;  cytological  methods,  365. 

HEROUARD,  Cucumaria,  481. 

HERTWIG,  silver  impregnation,  246, 
248;  maceration,  317,  484; 
Medusae  and  Actiniae,  317;  ova 
of  Triton,  342;  ova  of  Eana, 
343. 

HERXHEIMER,  plasma  fibrils,  367. 

HESCHL,  amyloid  degeneration,  210. 

HESSERT,  flagella,  496. 

HEURCK,  TAN,  mounting  medium,  281. 

HEYDENREICH,  amber  varnish,  292. 

HBYMONS,  embryology  of  Blattida, 
352. 

HICKSON,  eosin  and  haematoxylin, 
2G6;  maceration,  314;  eyes  of 
Musca,  467. 

HILL,  nerve  stain,  404;  Golgi's  im- 
pregnation, 417,  421. 

HIPPKL,  retina,  432. 

HIROTA,  egg  of  fowl,  339. 

Hirudinea,  472. 

His,  nitric  acid  fixation,  46 ;  impreg- 
nation, 242. 

HOCHSTETTER,  injection,  310. 

HOEHL,  bichromate  and  osmic  acid, 
44  ;  adenoid  tissue,  437. 

HOFER,  hydroxylamin,  15. 


516 


INDEX. 


The  numbers  refer  to  the  Payes. 


HOFFMANN,  vacuum  imbedding,  101 ; 
orientation,  104. 

HOFMANN'S  Griin,  222. 

HOGGAN,  histological  rings,  244 ;  iron 
stain,  260. 

HOLM,  liver,  457. 

Holothurioidea,  480. 

HOPEWELL-SMITH,  odontoblasts,  444. 

HOPKINS,  maceration,  317. 

Horn,  368. 

HOTER,  silver  impregnation,  246 ; 
gold  impregnation,  254;  mount- 
ing medium,  274;  carmine-gelatin 
injection,  301;  blue  gelatin  in- 
jection, 305 ;  yellow  gelatin  in- 
jection, 305,  306;  green  gelatin 
injection,  306;  shellac  injection, 
310;  oil-colour  injection,  310; 
mucin,  453. 

HOYEE,  jun.,  formol,  75;  Infusoria, 
494. 

HFBER,  medullated  nerve,  395;  Golgi's 
impregnation,  422. 

HUDSON,  Rotatoria,  473. 

HYATT,  shellac  imbedding,  136. 

Hydra,  485. 

Hydrate  of  chloral,  see  Chloral. 

Hydrochloric  acid,  for  decalcification, 
322,  324. 

Hydrochloric  acid  alcohol,  72. 

Hydrochloric  acid  carmine,  174. 

Hydrofluoric  acid,  326. 

Hydrogen  peroxide,  for  narcotisation, 
18;  for  bleaching,  34,  328. 

Hydroidea,  485. 

Hydroxylamin,  narcotisation,  15. 

Hypochlorite    of    potash,    321,    341; 

Hypochlorite  of  soda,  ,321. 

I. 

IDE,  double  imbedding,  134 ;  epithe- 
lium, 366. 

IGACUSCHI,  liver,  457. 

IIJIMA,  embryology  of  Planaria,  355. 

IKEDA,  section-fixing,  144. 

Imbedding,  defined,  7 ;  small  objects, 
332. 

Imbedding  methods,  90;  manipula- 
tions, 91,  98,  102,  103,  et  seq. ; 


trays,  thimbles,  91;  boxes,  92, 
93 ;  choice  of  method,  94 ;  in 
vacuo,  101 ;  paraffin,  96  et  seq.  / 
soap,  117,  478;  gelatin,  117; 
celloidin  (collodion^,  120. 
Impregnation,  defined,  241 ;  positive 
and  negative,  241 ;  primary  and 
secondary,  242. 

Impregnation      methods,     241 — 261 ; 
theory  of,  242  ;  silver,  243—249 ; 
gold,    249 — 258;     other    metals, 
258—261 ;  fats  (Altmann),  322. 
Indian  ink  injection,  310. 
:    Indifferent  liquids,  268—271. 
j   Indigen,  223 
Indigo  carmine,  264. 
Indigo  substitute,  223. 
|   Indulin,  223. 

;   Induliu-aurantia-eosin,  223. 
i   Infusoria,  490—495. 
Injections,  297—311;    gelatin,  298— 
306;     other    masses,    307—310; 
natural,  311,  471,   472;    of  Mol- 
lusca,  464  ;    of  Arthropods,  468  ; 
of  Hirudinea,  472. 
:   Insects,  see  Arthropoda. 
:    Intercellular    Bridges    and    canal?, 

366. 

Infra  mtam  staining,  156,  229. 
Inversion  stains,  226. 
lodate  of  sodium,  36. 
I   lodic  acid,  449. 
'   Iodide  of  palladium,  407. 
I   Iodine,  for    removing    sublimate,   55; 
for    fixing,   66 ;  for    mordanting, 
160 ;  for  hardening,  384 ;  LTJGOL'S 
solution,  66. 
Iodine  green,  222. 
Iodised  serum,  270,  271 ;  maceration 

in,  313. 

Iridium  chloride,  61. 
Iris,  375. 

Iron,  impregnations,  260. 
Iron  perchloride,  see  Perchloride. 
Iron-carmine,  170. 
Iron-hsematoxylin,  188—191,  502. 
Isopoda,  embryology,  354. 
ISEAEL,    Ehrlich-Biondi     stain,    217; 
acidophilous  mixture,  224 ;  orceiu, 
263. 


INDEX. 


o!7 


The  numbers  refer  to  the  Pages. 

IWANZOFF,  electric  organs,  434 
muscles  of  Holothurids,  481 
nematocysts  484. 


J. 

JACKSON,  cleariug,  80. 

JACOBS,  freezing  mass,  138. 

JACOBT,  bleu  de  Lyou,  225. 

JADASSOHN,  plasma  cells,  439. 

JAENICHEN,  Planaria,  480. 

Jaera,  ova,  354. 

JAGER,  glycerin  liquid,  278. 

JAKIMOVITCH,  silver  impregnation, 
247 ;  medullated  nerve,  395. 

JANDEB,  bleaching,  329. 

JANSSENS,  amyl  alcohol,  85;  iron 
haematoxylin,  191 ;  bleu  carmin, 
226. 

Japanese  section-fixing  method,  144. 

JAQUET,  leeches,  472. 

JAVELLE,  eau  de,  321. 

JELGERSITA,  anilin  blue-black,  409. 

JELINEK,  picric  acid,  67 ;  "  Stabilit," 
129. 

Jellies,  see  Glycerin. 

JENNEB,  blood,  448. 

JENNINGS,  Rotatoria,  355. 

JENSEN,  Infusoria,  491. 

JOEST,  Annelids,  472. 

JOHNSON,  Lindsay,  fixing  mixture,  44; 
cement  for  collodion  blocks,  129; 
sunning  metallic  solutions,  242; 
gold  impregnation,  255;  retina, 
431. 

JOHNSTON,  reconstruction,  332. 

JOHNSTONE-LAVIS,  section  grinding, 
137. 

JOLIET,  gum  imbedding,  135. 

JOBDAN,  clearing  agents,  80 ;  imbed- 
ding box,  93;  serial  sections,  147. 

JOSEPH,  silver  impregnation,  242;  in- 
jection, 307. 

JULIEN,  flagella,  496. 

JULIUSBURGEB,  stain  for  nerve-cells, 
392. 

JUNG,  K.,  microtomes,  etc.,  address, 
87 ;  knife-holders,  107. 

JUSCHTSCHKNKO,  Golgi's  impregna- 
tion, 421. 


K. 


KADTI,  soap  imbedding,  117. 
KAES,  nerve  gtuiii,  402. 

KAISER,  sublimate  solution,  57;  irlv- 
cerin  jelly,  118,  279;  Bismarck 
brown,  207;  nerve  stains,  402, 
410;  Acanthocephali,  474. 

KAISEBLING,  formol,  74. 

KALLIUS,  Golgi's  impregnation,  419, 
423. 

KABAWAIEW,  paraffin  stove,  101  ; 
Protozoa,  494. 

KASTSCHENKO,  reconstruction,  332. 

KEMP,  blood-platelets,  452. 

KENT,  fixative,  66. 

KENYON,  phospho-molybdic  haema- 
toxylin, 191 ;  Pauropoda,  466  ; 
brain  of  bees,  468. 

Keratohyalin,  366. 

Kernschwarz,  262. 

Kidney,  458. 

Killing,  generalities,  11 ;  various  pro- 
cesses, 11—18. 

KINGSLEY,  embryology  of  Limulus, 
353. 

KIONKA,  egg  of  fowl,  339. 

KISHINOUYE,  embryology  of  Araneida, 
352;  of  Limulus,  353. 

KIZEB,  blood,  448. 

KLEBS,  glycerin  jelly,  118. 

KLEIN,  chromic  acid,  39 ;  cornea,  369. 

KLEINENBEBG,  picro-sulphuric  acid, 
67;  haematoxylin,  186;  colo- 
phonium,  285 ;  Lopadorhyncus, 
354. 

KLINCKOWSTBOEM,  Prosthecereeus, 
480. 

KNAUEB,  cleaning  slides,  500. 

Knife  position,  104—107. 

Knife-holders,  107. 

Knife- warmers,  110,  501. 

Knives,  microtome,  105. 

KOCH,  TON,  section  method,  136. 

KOFOID,  embryology  of  Gastropoda, 
348. 

KOGANEI,  iris,  375. 

KOHLEB,  Taeniae,  478. 

KOLLIKEB,  ova  of  rabbit,  334,  336; 
bone,  445. 

KOLLMANN,  fixing  ova,  344. 


518 


INDEX. 


The  numbers  refer  to  the  Pages. 


KOLOSSOW,  clarifying  osmic  acid,  32  ; 
osmic  mixtures,  36 ;  gold  method, 
255 ;  osmic  acid  stain,  260 ;  epi- 
thelium, 366;  Grolgi's  impregna- 
tion, 421. 

KOLSTEE,  gastric  glands,  456. 

KOPPEN,  elastic  tissue,  441. 

KOPSCH,  embryology  of  Salmonidse, 
345;  Golgi's  impregnation,  420; 
eyes  of  Cephalopods,  463. 

KOBOTNEFF,  Siphonophora,  13. 

KOESCHELT,  embryology  of  Loligo, 
348;  of  Ophryotrocha,  354; 
Protozoa,  492. 

KOSSINSZI,  stain,  224. 

KOSTANECZI  and  SIEDLECKI,  ova  of 
Ascaris,  356  ;  sublimate  mixture, 
58. 

KOSTANECKI  and  WIEEZEJSZI,  ova  of 
Physa,  349. 

KOTLAEEWSZY,  ganglion  cells,  382. 

KOWALEWSZY,  fishes,  embryology, 
345. 

KBAUSE,  Ehrlich-Biondi  stain,  215, 
216 ;  thiophen  green,  223 ;  retina, 
432,  433;  salivary  glands,  456; 
corpuscles  of,  369. 

KBATTSS,  silver  impregnation,  247 ; 
hardening  nervous  tissue,  387. 

Kreasote,  refractive  index,  81 ;  for 
clearing,  85. 

KBOHNTHAL,  lead  impregnation,  427. 

KBOMAYEE,  plasma  fibrils,  367 ;  con- 
nective tissue,  436. 

KBONECZEB'S  serum,  271. 

KBONIG,  cement,  292. 

KBYSINSKY,  photoxylin,  121. 

KUHNE,  H.,  freezing  method,  139. 

KUHNE,  W.,  maceration,  317 ;  diges- 
tion, 320. 

KUHNT,  retina,  432. 

KUZENTHAL,  narcotisation,  15,  470; 
blood-vessels,  471 ;  intestine  of 
Lumlricus,  471. 

KTTLTSCHITZKY,  preservation,  5 ;  fixing 
liquids,  52;  double  imbedding, 
134;  tactile  corpuscles,  368; 
hsematoxylin  nerve  stain,  402; 
neuroglia,  429;  elastic  tissue, 
441 ;  mucus  cells,  455 ;  spleen,  457. 


KTTPFFEE,      embryological      methods, 
341 ;     axis-cylinder     stain,    393 
liver,  457. 

KUSKOW,  digestion,  319. 

L. 

LABAEEAQTJE,  eau  de,  321. 

Labels,  gum  for,  501. 

Labyrinth,  326,  434. 

LACHI,  formol  for  nerve  tissue,  386 

Lactic  acid,  325. 

Lamellibranchiata,  461—464;  embry- 
ology, 349 

LANDOIS,  impregnations,  sulphides, 
261 ;  maceration,  315. 

LANG,  liquid  of,  57. 

LANGEBHANS,  mounting  medium,  274 ; 
tactile  corpuscles,  368. 

LANZESTEE  and  BOUBNE,  eyes  of 
Limulus,  467. 

LANSBEEG,  Protozoa,  493. 

Larynx,  456. 

LASLETT,  nerve  stain,  402. 

LATTTEBBOEN,  Protozoa,  494. 

LATDOWSZY,  formol  mixture,  76; 
methylen  blue,  236 ;  chloral  pre- 
servative solution,  272  ;  saudarac 
for  mounting,  287  ;  cochlea,  434 ; 
blood,  449 ;  maceration,  314. 

LAWBENCE,  glycerin  jelly,  278. 

Lead  acetate,  382. 

Lead  chromate,  impregnation,  260. 

Lead  sulphide,  impregnation,  261,427. 

LEBEB,  impregnations,  260;  retina, 
432. 

LECAILLON,  ova  of  Coleoptera,  352. 

LEE,  A.  B.,  preservation  of  material, 
5,  6;  lemon  juice  for  fixing,  12; 
narcotisation,  18 ;  keeping  osmic 
acid,  31 ;  making  up  chromo-aceto- 
osmic,  42 ;  nitric  acid  for  fixing, 
46;  sublimate  solution,  54 ;  picro- 
acetic  acid,  67  ;  formol,  75 — 77  ; 
cedar  oil,  81,  82,  97;  oil  of 
turpentine,  84;  mounting  sections 
in  balsam,  85 ;  paraffin  masses, 
115;  celloidin  imbedding,  122, 
125,  133 ;  Mayer's  albumen,  143, 
144,  145,  147  ;  infra  vitam  stain- 
ing, 157 ;  saf  ranin,  203  ;  toluidin 


INDEX. 


519 


The  numbers  refer  to  the  Pages. 


blue,   208;  orange  G,  213;  Sau- 

refuchsin,  215 ;    osmic   acid   and 

pyrogallol,      258,      259;      Kern- 

schwarz,  262 ;  glycerin  liquid,  277  ; 

colophcnium  solution,  285 ;  cedar 

oil  for  mounting,  286  ;  paper  cell 

mounting  method,  289  ;  cytologi- 

cal  methods,  358  et  seq. ;  Alcyo- 

naria,      485;    Hirudinea,      472; 

Nemertina,    476,    477 ;    sponges, 

489. 

LEGAL,  picro-alum -carmine,  169. 
LEGBOS,  silver  impregnation,  248. 
Lemon  juice  for  fixing,  12 
LENDENTELD,  vox,  sponges,  489. 
LENHOSSEK,      TON,     ccerulein,     223; 

thionin,  391;  toluidin  blue,  392; 

eyes  of  Cephalopods,  463 ;  nerves 

of  Annelids,  471. 
LENNOX,  retina,  432. 
LENS,  crystalline,  370. 
Lepidoptera,  embryology,  351. 
LEPKOWSKY,  teeth,  444. 
LEUCKHABT,  imbedding  boxes,  92. 
Leucocytes,  451,  452. 
Levulose  for  mounting,  275. 
LEWIS,  BEVAN,  anilin  blue-black,  225, 

409  ;  hardening  brain,  379,  384. 
LEWIS,  M.,  nerves  of  Annelids,  471. 
Lichtgriin,  208,  222. 
LIEBERMANN,  carmine,  163 
Light,  action  on  alcohol  with  chromic 

material,   39 ;  on   metallic    salts, 

242. 

Light  green,  222. 
LILIENFELD,  blood-plates,  452. 
LILLIB,  embryology  of   Unto,  349. 
Limulus,  embryology,  353. 
LINDSAY  JOHNSON,  nee  JOHNSON. 
Linseed  oil  injection,  311. 
Liquid    of     Miiller,    of     Erlicki,    of 

Merkel,  etc.,  see  the  names  of  the 

respective  authors. 
Liquidambar,  287. 
Liquor  ferri  sulphurici  oxidati,  188. 
LIST,  methyl  green  and   eosin,   221 ; 

haematoxylin     and     eosin,    266 ; 

goblet- eel  Is,  456  ;  Coccidse,  467  ; 

Actinida,  484. 
Liver,  457. 


LIVINI,  elastic  tissue,  441. 

Lo  BIANCO,  tobacco  narcotisation,  12  ; 
alcohol  narcotisation,  14 ;  chloral 
narcotisation,  15 ;  poisoning  me- 
thod, 16  ;  chromo-acetic  acid,  40  ; 
chromo-osmic  acid,  40;  osmic  acid 
and  bichromate,  44 ;  sublimate 
solution,  55;  chrotnic  sublimate, 
59;  acid  alcohol,  73  ;  methods  for 
marine  animals,  459  et  *eq. ; 
Actinida,  484 ;  Alcyonaria,  485  ; 
Asteroidea,  482 ;  Brachiopoda, 
461;  Bryozoa,  460;  Chaetopoda, 
470;  Crinoidea,  482  ;  Ctenophora, 
489  ;  Echinoidea,  482 ;  Entero- 
pneusta,  469;  Gastropoda,  462; 
Gephyrea,  473 ;  Holothurioidea, 
480,  481 ;  Lamellibranchs,  461 ; 
Medusae,  486 ;  Neniatoda,  475  ; 
Nemertina,  476;  Ophiuridea,  482  ; 
Protozoa,  493;  Siphonophora,  488 ; 
Trematodes,  478;  Tunicata,  459, 
460 ;  Turbellaria,  480 ;  Zoantharia, 
485. 

LOCKE,  salt  solution,  269. 

LOCY,  embryology  of  Araneida,  353. 

LOEWENTHAL,  liquid  of  Erlicki,  52. 

LOEWY,  epidermis,  366. 

LOFFLEB,  stain  for  flagella,  495. 

LoiSEL,   intra    vitam   staining,    158 ; 
Congo  red,  220 ;  neutral  red,  220. 

LONGHI,  Protozoa,  494. 

LONGWOETH,  corpuscles    of    Krause, 
369. 

LONNBEBG,  Tri&nophorus,  477. 

Looss,  eau  de  Labarraque,  321 ;  Nema- 
todes,  475  ;  Bilharzia,  478. 

LOED,  Nissl's  stain,  390. 

LOWE  lens,  370. 

LOWIT,  gold  method,  252 ;  blood,  449. 

LUGOL,  iodine  solution,  66. 

LUITHLBN  and  SOBGO,    Nissl's  stain, 
391. 

LUSTGABTEN,  Victoria  blue,  206. 

LUXENBFBG,  stain  for  nerve-cells,  392. 

Lysol,  for  maceration,  318. 

M. 

MAAS,  carmine  and  malachite  green, 
265 ;  larvae  of  sponges,  490. 


520 


INDEX. 


The  numbers  refer  to  the  Pages. 


MACALLUM,  caniiitie  aud  indigo-car- 
mine, 264. 

MAC  BRIDE,  AmpUura,  483. 

Maceration,  312 — 319;  maceration  of 
epithelium,  464. 

Magdala  red,  207. 

Magenta,  207. 

Magenta  S,  215. 

MAGINI,  zinc  impregnation,  427. 

Magnesia-carmine,  171. 

Magnesium  chloride,  narcotisation, 
16;  Magnesium  sulphate,  nar- 
cotisation, ]  6. 

MAHRENTHAL,  VON,  osmic  acid  stain, 
260. 

Malachite  green,  222,  265. 

MALASSEZ,  ammonia-carmine,  171 ; 
salt  solution,  269. 

MALLORY,  phospho-molybdic  hsema- 
toxylin,  191 ;  phospho-tungstic 
haematoxylin,  192 ;  Weigert's 
neuroglia  stain,  429. 

Mammalia,  embryology,  333 — 337. 

Manchester  brown,  210. 

MANFREDI,  gold  method,  255. 

Manganese  chloride,  269. 

MANN,  picro-sublimate,  59;  osmio- 
subliraate,  59;  serial  sections, 
145  ;  fixing  nerve-centres,  379  ; 
hsematein  stain^'  185;  toluidin 
blue,  208 ;  Wasserblau,  225. 

MAECANO,  blood,  448. 

MARCHESINI,  medullated  nerve,  395. 

MAECHI,  corpuscles  of  Golgi,  373 ; 
degenerate  nerves,  404 ;  mucus  of 
Gastropoda,  462. 

MAECUS,  formol  for  spinal  cord,  386. 

MAEINA,  fixing  nervous  system,  387. 

Marine  animals,  precautions  in  pre- 
paring, 27 ;  silver  impregnation, 
248 ;  gold  impregnation,  258. 

Marine  glue,  291. 

MARX,  collodionising  sections,  111. 

MABPMANN,  fluorides,  61. 

MAESCHALKO,  plasma  cells,  439. 

MAESH,  carmine  and  indigo-carmine, 
264;  gelatin  cement,  289. 

MAETIN,  benzo-azurin,  207,  226. 

MAETINOTTI,  C.,  elastic  tissue,  441. 

MARTINOTTI,  G.,  damar,  284;   anilin 


blue-black,  409 ;  picro-nigrosin, 
409 ;  elastic  tissue,  440. 

MAETINOTTI  and  RESEGOTTI,  safnmin, 
204. 

MASON,  nervous  system  of  reptiles, 
387. 

Mastzellen,  437—439. 

MATSCHINSKY,  bone,  443. 

MAUEICE  and  SCHTJLGIN,  bleu  de  Lyon, 
225,  265. 

MAYER,  P.,  principles  of  technique, 
10 ;  preparing  marine  animals, 
28 ;  bleaching  osmic  objects,  35  ; 
washing  out  chromic  objects,  37 ; 
chromic  acid  and  alcohol,  39; 
liquid  of  Perenyi,  47 ;  washing 
sublimate  material,  55 ;  picro- 
sulphuric  acid,  68;  picro-nitric, 
68;  picro-hydrochloric,  68;  tt-sts 
for  alcohol,  70 ;  acid  alcohol,  72 ; 
paraffin  imbedding,  92,  93,  99, 
116;  water-bath,  100, 102;  gelatin 
imbedding,  117 ;  serial  sections, 
water  method,  140;  albumen 
method,  143, 147 ;  shellac  method, 
146 ;  theory  of  staining,  155 ; 
staining  with  carmine,  163 — 166  ; 
staining  with  cochineal,  165 ; 
carmalum,  167;  aluminium  chlo- 
ride carmine  stain,  167 ;  alum- 
carmine,  168 ;  PAETSCH'S  cochi- 
neal, 168 ;  magnesia-carmine,  171 ; 
picro-carmine,  171;  picro-mag- 
nesia-carmine,  172;  borax-carmine, 
172,  173 ;  paracarmine,  173 ; 
.  hydrochloric  acid  carmine,  174 ; 
alcoholic  cochineal,  174 — 176; 
theory  of  hsematoxylin  staining, 
177—179;  h£ematemri79;  hsema- 
teate  of  ammonia,  180 ;  bluing 
hsematein  stains,  180, 181 ;  haema- 
lum,  182,  183 ;  glycheemalum, 
184;  Ehrlich's  haernatoxylin,  185  ; 
hsemacalcium,  185 ;  methyl  violet, 
208;  methyl  green,  208;  iodine 
green,  223 ;  Bismarck  brown,  211 ; 
Kernschwarz,  262  ;  Brazilin,  263  ; 
carmine  and  indigo-carmine,  264  ; 
hajmalum  and  indigo-carmine, 
264;  mucicarmine,  454;  muchse- 


INDEX. 


521 


The  numbers  refer  to  the  Pages. 


matein,    455;   balsam,   282,   283; 

Venice   turpentine,   286;    decalci- 

tication,  324,  325  ;  desilicification, 

326;    bleaching,   327;    injection, 

309 ;  mucus,  454,  455. 
MAYER,  P.,  ANDRES,  and  GIESBUECHT, 

section-stretcher,  109. 
MAYEB,    P.,    and    SCHOEBEL,    knife- 
holders,  107. 
MAYEB,   S.,   neutral  red,  220;  violet 

B,  226;  methylen  blue,  233,  235, 

239  ;  connective  tissue,  435. 
MAYSEL,  Bismarck  brown,  210. 
McCBOBlB,    night-blue    for    flagella, 

496. 

McMuRRicn,  ova  of  Jaera,  354. 
Medullated  nerve  structure,  393 — 395 ; 

stains  for,  396 — 408. 
Medusae,  486,  487. 
MKHSEBT,     embryological      methods, 

341. 
MEISENHEIMEB,  embryology  of  Limax, 

348. 

MEISSNEB,  corpuscles  of,  252,  369. 
MELNIKOFF-RASVEDENKOFF,     formol, 

74. 

Menthol  for  narcotisation,  15. 
MEBCIEB,  nerve  stain,  404. 
Mercuric  mixtures,  57  et  seq.,  and  see 

Sublimate. 
Mercury,    bichloride,    see    Sublimate; 

biniodide,  280. 

MEEK,  liquid  of  Flemming,  43. 
MERKEL,  chromo-plat'mic  mixture,  48; 

carmine  and  indigo-carmine,  264. 
MEBKEL    and    KEAUSE,   molybdenum 

impregnation,  261. 
MERKEL  and  SCHIEFFEEDECKEE,  eel- 

loidin  imbeddiug,  121. 
Metagelatin,  298. 

Metallic  salts,  action  of  light  on,  242. 
Metallic  stains,  241—261. 
Metanil  yellow,  214. 
METCALP,  embryology  of  Chiton,  349. 
Methyl  alcohol,  tor  narcotisation,  14; 

refractive  index,  81. 
Methyl  blue,  225. 
Methyl  green,  208. 
Methyl  green  and  eosin,  221. 
Methyl  sal  icy  late,  86. 


Methyl  violet,  207;  test  for,  208; 
progressive  stain,  211. 

Methyl  violet  B,  226. 

Methylal,  for  dehydration,  5. 

Methylanilin  green,  208. 

Methylanilin  violet,  211. 

Methylated  spirit,  497. 

Mt-thylen  blue,  chemistry  of,  228  ;  uses 
of,  229  ;  for  intra  vitam  staining, 
229;  for  central  nervous  s\  stern, 
430;  for  impregnation,  228—239  ; 
generalities,  229 — 232  ;  staining 
nervous  tissue,  230—239,  389 — 
391, 393, 430;  staining  by  injection 
or  immersion,  232 ;  the  solutions 
employed,  232 ;  preservation  ot' 
the  preparations,  235 — 239;  im- 
pregnation of  epithelia,  etc.,  239. 

Methyleu  blue,  polychromatic,  228. 

Methylen  blue  and  eosin,  222. 

Methylen  blue  and  erythrosin,  391. 

Methylen  blue  and  fuchsin,  226. 

Methylen  red,  228. 

MEYEE,  E.,  celloidin  sections,  132. 

MEYEE,  F.,  salicylic  vinegar,  274. 

MEYEE,  SEMI,  methyleu  blue  for 
nerve  centres,  430. 

MIBELLI,  elastic  tissue,  440. 

MICHAELIS,  ova  of  Triton,  343. 

Micro-chemistry  of  the  cell,  359. 

Microtome  knives,  105,  106;  micro- 
tomes, 87—89,  104. 

MIGTJLA,  glycerised  serum,  271. 

MILLEE,  caoutchouc  cement,  289,  290; 
injection,  306. 

MINOT,  microtome,  89;  haematoxylin 
stains,  192;  celloidin  sections, 
131. 

Miracidia,  356. 

MITEOPHANOW,  double  imbedding, 
135;  Wasserblau,  225;  nerve 
stain,  403. 

MITSTJKUBI,  embryology  of  tortoise, 
340. 

MOBIUS,  maceration,  316. 

MOEBNEB,  cartilage,  446. 

MOLESCHOTT,  maceration,  314. 

MoLESCHOTTand  PJSO  BOBME,  macera- 
tion, 313. 

MOLLEB,  picro-Saurefuchsin,  215,  267. 


522 


INDEX. 


The  numbers  refer  to  the  Pages. 


Mollusca,  461  et  seq. ;  embryology, 
347—349. 

Molluscoidu,  460. 

Molybdate  of  ammonium,  impregna- 
tion, 261. 

MONCKEBERG  and  BETHE,  treatment 
of  osmic  material,  34,  328 ;  peri- 
pheral nerves,  410. 

Monobromide  of  naphthalin,  81,  281. 

MONTGOMERY,  Nemertina,  477. 

MONTI,  copper  impregnation,  427. 

MOORE,  A.  Y.,  blood,  450. 

MOORE,  V.  A.,  freezing  method,  139. 

Mordants,  159  et  seq. 

MORGAN,  embryology  of  Amphibia, 
342, 343 ;  embryology  of  Ascidians, 
346  ;  embryology  of  Periplaneta, 
350. 

MORTON,  flagella,  496. 

MOSELEY,  shell,  464. 

Motor  nerve-endings,  371  et  seq. 

Mounting  in  fluids,  289. 

Mounting  media,  see  Examination  and 
Preservation. 

Muchaematein,  455. 

Mucicarmine,  454. 

Mucicarminic  acid,  455. 

Mucin,  453—456. 

Mucus,  removal  from  Gastropoda,  462. 

Mucus  cells,  453—456. 

MUIR,  blood,  447. 

MIJLLER,  solution  of,  51 ;  for  macera- 
tion, 316 ;  for  injection,  309. 

MULLER,  C.  F.,  silver  impregnation, 
247. 

MULLER,  G.  W.,  Ostracoda,  466. 

MUNDER,  address,  162. 

MTJNSON,  chloral  hydrate,  272. 

Muscle,  smooth,  374 — 376. 

Muscle  and  tendon,  371 — 376. 

Muscle  cells,  371. 

Myelin  stains,  396—408. 

Myzostoma,  469. 

N. 

NABIAS,  R.  DE,  nervous  system  of  Pul- 

monata,  463. 
Nails,  368. 

NANSEN,  maceration,  316. 
Naphtha,  for  imbedding,  96. 


Naphthalin,  monobromide  of,  81,  281. 

Naphthalin  red,  207. 

Narcotisation,  12  et  seq. 

NATHTJSIUS,  VON,  horn,  368. 

Natural  injections,  311. 

NEALEY,  bone  and  teeth,  444. 

NEELSEN  and  SCHIEFFERDECKKR, 
clearing  agents,  80;  origanum 
oil,  83  ;  sandal-wood  oil,  84. 

Negative  impregnation,  241. 

Nematocysts,  484. 

Nematoda,  475  ;  embryology,  356. 

Nemertina,  476. 

NepJielis,  18. 

Nerve-cells,  389—393. 

Nerve-endings  in  muscle  and  tendon,. 
371—376;  in  skin  and  others,  368 
— 370;    and    see   Methylen    blue 
and  Neurological  methods. 
|  Nerve-fibres,  structure,  393—395. 
|   Nervous  centres  of  reptiles,  fishes,  and 
Amphibia,    387;    of    Gastropoda,. 
463 ;  of  Arthropods,  468;  and  see 
Neurological  methods. 

Nervous  system,  see  Neurological 
methods. 

NESTEROFFSKY,  gold  method,  255. 

NEUMAYER,  knife-wedges,  107. 

Neuroceratin,  393. 

Neuro- fibrils,  394,  502. 

Neuroglia,  428 — 430. 

Neurological  methods,  377;  introduc- 
tion and  hardening  methods,  37T 
—389;  cytological  methods,  389 
— 395;  myelin  stains,  396 — 406; 
myelin  and  axis-cylinder  ditto,, 
407,  408  ;  axis-cylinder  and  proto- 
plasm ditto,  409—430;  retina,, 
inner  ear,  etc.,  431. 

Neutral  balsam,  283. 

Neutral  chromate  of  ammonia,  52. 

Neutral  differentiation  or  extraction,, 
198. 

"  Neutral  "  dyes,  193. 

Neutral  red,  220 ;  for  nerve-cells,  392  ; 
for  mucus  cells,  455. 

Neutralisation   of    carmine    solutions,, 

•301. 

NIAS,  cleaning  slides,  500. 
NICOLAS,  gelatin  imbedding,  119. 


INDEX. 


523 


The  numbers  refer  to  the  Pages. 
Nicotiu  for  narcotisation,  13.  cloves,  9,  81,  9.6,  199;  lemons,  81  ; 


NIESSING,  fixing  liquids,  362. 

NIETZKI,  carrainic  acid,  164;  haeraa- 
teiu,  177. 

Xigranilin,  226. 

Nigrosin,  as  a  chromatin  stain,  207 ; 
as  a  plasma  stain,  223. 

NIKIFOBOW,  Ehrlich's  acidophilous 
mixture,  224;  myelin  stain,  408. 

XISSL,  stain  for  nerve-cells,  389. 

Nitrate  of  silver  impregnation,  243 — 
249 ;  generalities,  243 ;  solutions, 
245  ;  reduction,  247 ;  af  ter-black- 
ening,  248 ;  marine  animals,  248  ; 
vulcanite  rings  for,  244  ;  and  see 
GOLGI. 

Nitrate  of  uranium,  36. 

Nitric  acid,  for  fixing  and  hardening, 
46,  382  ;  for  maceration,  317 ;  for 
•  corrosion,  321;  for  decalcification, 
323,  324 ;  for  bleaching,  329. 

Nitrite  of  amyl,  297. 

NOACK,  orientation,  108. 

NOCHT,  methylen  blue  and  red,  228. 

Noir  Colin,  226. 

NOLL,  mounting  medium,  274;  cor- 
rosion, 321. 

NOBDMANN,  plasma  cells,  438. 

Normal  salt  solution,  269. 

NOBBIS  and  SHAKESPEABE,  carmine 
and  indigo-carmine,  264. 

NOWAK,  water-hath,  114. 

Nucleoli,  365. 

NUSBATJM,  serial  sections,  140,  142. 

0. 

OBEBSTEINEB,  hardening  nerve- 
centres,  382. 

OBBEGIA,  serial  sections,  151 ;  Golgi's 
impregnation,  423. 

ODENIUS,  maceration,  318. 

OHLMACHEB,  sublimate  alcohol,  65; 
section  fixing,  144;  mordanting 
with  formalin,  200 ;  safranin 
artefacts,  204  ;  picro-Saurefuch- 
sin,  215  ;  myelin  stain,  408. 

Oil,  of  aniseed,  81, 189 ;  bergamot,  81, 
83,  97;  cajeput,  84  ;  cedar,  6,  9, 
81,  97,  286 ;  cinnamon,  81,  83 ; 


origanum,  83;  sandal-wood,  84; 
thyme,  84 ;  turpentine,  81,  84,  96, 
286 ;  and  see  Clearing  agenta. 

Olive  oil,  refraction,  81. 

Ophiuriden,  482. 

OPPEL,  gastric  glands,  456;  liver  and 
spleen,  457. 

OPPITZ,  silver  impregnation,  247. 

Optical  differentiation,  23,  41. 

Orange  G,  213,  214. 

Orcein,  Israel's  method,  263 ;  Unna's, 
436,  441. 

Orchella,  263. 

Orchestia,  ova,  354. 

Orientation,  in  paraffin,  103,  104;  in 
celloidin,  124;  of  blocks,  107, 
108,  501. 

Origanum  oil,  83. 

Orseille,  263. 

OBTH,  "  Formol-Muller,"  76,  387. 

Osmic  acid,  generalities,  23,  31 ;  how 
to  keep,  31 ;  regeneration  of,  32 ; 
fixation  with, 33;  after-treatment, 
34 ;  characters  of  the  fixation, 
35;  blackeuing  of  fat,  36;  mix- 
tures, 36,  40,  et  seq. ;  sublimate 
mixtures,  59 ;  picric  mixtures,  69 ; 
stains  with  pyrogallol,  pyrolig- 
neous  acid  or  tannin,  258 — 260; 
stains  for  metlullated  nerve,  404 
—406. 

Osmicated  cells,  35. 

Osmic-bichromic  mixtures,  44. 

Osmio-sublimate  mixtures,  59. 

Osmium,  see  Osmic  acid. 

Osmium-carmine,  479. 

Osmosis,  to  avoid,  4. 

Ostracoda,  466. 

Otocyst  of  Mysis,  469. 

Ova,  see  Embryological  methods. 

Ovens,  100. 

OYEBTON,  bleaching,  34,  38;  fixing 
with  iodine,  66 ;  fixing  Infusoria, 
53. 

OVIATT  and  SABGENT,  injecting,  298. 

Oxalic  acid,  for  maceratio,n  318. 

Oxygenated  water,  for  narcotisation, 
18;  for  bleaching,  34,328. 


524 


INDEX. 


The  numbers  refer  to  the  Pages. 


P. 


PACINI,  preservative  liquids,  273. 

PAL,  nerve  stain,  400;  Golgi's  subli- 
mate method,  426. 

PALADINO,  nerve  stain,  407. 

Palladium  chloride,  for  fixing,  61 ; 
staining,  260 ;  decalcifying,  323. 

Palladium  iodide,  407. 

Pancreatin  digestion  fluid,  319. 

PANETH,  hsematoxylin,  398)  goblet- 
cells,  456. 

PANSCH,  starch  injection,  311. 

Paper  cell  mounting  method,  289. 

Paper  trays  and  thimbles,  91. 

Paracarmine,  173. 

Paraffin,  for  preserving  material,  6  ; 
solvents  of,  96,  97;  imbedding  in, 
91 — 117 ;  orienting  in,  104 ;  cut- 
ting, 108—113;  ribbons,  112; 
coating  blocks  of,  113;  electrifica- 
tion of,  113 ;  masses  recommended, 
115,116  ;  mounting  sections,  140 ; 
cement,  292. 

Paraffinum  liquidum,  81. 

PAEKEE,  dehydration,  5 ;  methylen 
blue,  237 ;  turpentine  cement, 
291;  bleaching,  329;  eyes  of 
Arthropods,  467. 

PAEKEE  and  FLOYD,  formol  for  brain, 
386. 

Parma  blue,  224. 

PAETSCH,  cochineal,  168. 

PASSAEGE  and  KEOSING,  elastic  tissue, 
441. 

PATTEN,  orientation  in  paraffin,  103; 
embryology  of  Blattida,  352;  eyes 
of  Lamellibranchs,  463 ;  macera- 
tion of  Mollusca,  465. 

PATTLSEN,  goblet-cells,  456. 

Pauropoda,  466. 

PEABODY,  methylen  blue,  238. 

Pelagic  ova,  346. 

Pentacrinus ,  483. 

Pepsin  digestion  fluids,  319. 

Perchloride  of  iron,  for  fixing,  61 ;  for 
staining,  260. 

PEEEMESCHKO,  Iarva3  of  Amphibia, 
358. 

PEEENYI,  chromo-nitric  acid,  46. 

Permanganate  of  potash,  for  bleaching, 


34,  328;  for  mordanting,  160, 
200;  for  maceration,  316. 

Perophora,  347. 

Peroxide  of  hydrogen, for  narcotisation, 
18;  for  bleaching,  34,  328. 

Peroxide  of  sodium,  34,  328. 

PEEEIEE,  Lumbricus,  470. 

Perruthenic  acid,  456. 

Petroleum-ether,  97. 

PFEIFFEE  VON  WELLHEIM,  iron-car- 
mine, 170. 

PFISTEE,  hardening  nerve-centres,  381. 

PFITZNER,  safranin,  203;  damar  solu- 
tion, 284 ;  Protozoa,  492. 

Phalangidn,  embryology,  352. 

Pheuicienue,  La,  210. 

Phenylen  brown,  210. 

PHILIPPSON,  epidermis,  366. 

Phloroglucin,  325. 

Phloxin,  221. 

Phospho-molybdic  hsematoxylin.  191. 

Phosphoric  acid,  323,  325. 

Phospho-tungstic  hsernatoxylin,  192. 

Photoxylin  for  imbedding,  121. 

Physa,  ova,  349. 

Physiological  salt  solution,  269. 

PIANESE,  formic  acid  carmine,  170; 
methylen  blue  and  eosin,  222 ; 
carmine  and  picro-nigrosin,  265. 

Picric  acid,  fixation  with,  66 — 69; 
solubility,  66;  washing  out,  67; 
as  a  plasma  stain,  212;  for  decal- 
cification,  325. 

Picric  alcohol,  69,  316. 

Picro-acetic  acid,  67. 

Picro-aluni-carmine,  169. 

Picro-carmine,  171,  172. 

Picro-chromic  acid,  69. 

Picro-hydrochloric  acid,  68,  325. 

Picro-indigo-carniine,  265. 

Picro-maguesia-carmine,  172. 

Picro-nigrosin,  265,  409. 

Picro-nitric  acid,  68,  325. 

Picro-niti*o-chromic  acid,  69. 

Picro-osmic  acid,  69. 

Picro-platinic  mixtures,  69. 

Picro-Saurefuchsin,  215;  with  ha3ina- 
toxylin,  267. 

Picro-sublimate  mixtures,  58. 

Ficro-sulphuric  acid,  67. 


INDEX. 


The  numbers  refer  to  the  Pages. 
PICTET,  examination  liquid,  269.  Preservation  of  material,  5,  6. 


Pigment,  removal  of,  467. 
Pigment  spots,  artificial,  383. 
PIXT>-ER,  Taniae,  477. 
Pisces,  embryology,  344. 
PlSENTl,  alum-carmine,  168. 
Pizox,  gemmation  of  Ascidians,  346. 
Plasma  cells,  437—439. 
Plasma  fibrils,  367. 

Plasma  stains,  defined,  154,  212;  the 
coal-tar,  212—227;  cytological, 
364. 

Plastic  reconstruction  of  sections,  332. 
Platino-aceto-osmic  acid,  45,  362. 
Platino-sublimate  mixture,  331. 
Platinum    chloride,  60 ;  mixtures,  44, 

45,  48,  331. 
PLAINER,  medullated  nerve,  61,  394; 

Kernschwarz,  262. 
PLESCHKO,  methylen  blue,  239. 
PLESSIS,  Dr,  see  Du  PLESSIS. 
Pli'tei's,  483. 
PODWYSSOZKI,     fixing     mixture,   43; 

safrauin,  204.      - 
Poisoning,  16  et  seq. 
POLAILLON,  iron  impregnation,  260. 
Polari*ation,  for  myelin,  406. 
POLITZER,  inner  ear,  434. 
POLUMORDWINOW,  stain     for    nerve- 
cells,  392. 

Polychaeta,  embryology,  354. 
Polychromatic  methylen  blue,  228. 
POLZAM,  soap  imbedding,  117. 
POPOW,  neuroglia,  430. 
Porifera,  489,  490. 
Positive  impregnation,  241. 
Potash,     for     maceration,     314 ;     for 
corrosion,    321 ;    acetate    of,   see 
Acetate ;   bichromate  of,   see   Bi- 
chromate;    hypochlorite    of,    see 
Hypochlorite ;    permanganate   of, 
see  Permanganate. 

Potassium  bichromate,  see  Bichromate. 
Potassium  ferricyanide,  for  bleaching, 

35. 

Potassium    permanganate,  for  bleach- 
ing, 34,  328 ;  as  a  mordant,  160, 
200 ;  for  maceration,  316. 
POUCHET,  bleaching,  328. 
PRENAXT,  saf ranin,  204 ;  cochlea,  433. 


Preservative   media,  see   Examination 

and  Preservation. 
PEETEK,  star-fishes,  14. 
Prickle-cells,  366. 
Primerose,  221. 
PBINCE,  blood,  451. 
PXINGLE,  vacuum  imbedding,  101. 
PBITCHABD,  chromic  acid  mixture,  39  ; 

reducing  liquid,  255  ;  cochlea,  434. 
Progressive  staining,  154,  195. 
Protozoa,  490—495. 
PBOWAZEK,  neutral  red,  220. 
PBTTDDEN,  hannatoxyliu,  184. 
Prussian     blue,    impregnation,     260; 

injections,    299,    303—305,    308, 

309;  soluble,  303. 
PBZESMYCKI,  Protozoa,  492. 
PUBCELL,  eyes  of  Phalangida,  467. 
Purpurin,  264. 
Pyridin,  73,  379. 
Pyrogallate  of  iron  stain,  260. 
Pyro^rallate  of  osmium  stain,  258. 
Pyroligrneous  acid  carmine,  170. 
Pyroligneous  acid  haematoxylin,  187. 
Pyrosin,  221. 
Pyrosoma,  460. 

Q. 

QUERTAIN,    DE,    fixation    of    nervous 

tissue,  379. 

Quieting  Infusoria,  etc.,  491. 
Quinole'in,  223. 


R. 

Rabbit,  embryology,  333—336. 

RABL,  chromo-formic  acid,  40;  picro- 
sublimate,  58  ;  platinum  chloride, 
60  ;  paraffin  sections,  112,  116 ; 
serial  sections,  145 ;  cochineal, 
169;  htematoxylin  and  safranin, 
267  ;  embryological  methods,  331, 
343. 

RABL-RUCZHABD,  embryology  of  Sal- 
monida?,  345. 

RAFPAELE,  pelagic  ova,  34C. 

Raja,  434. 

RAMON  Y  CAJAL,  see  CAJAL. 

RANVIEB,   osmic    acid,   36 ;    chromic 


526 


INDEX. 


The  numbers  refer  to  the  Pages. 


acid,  38;  one-third  alcohol,  71; 
absolute  alcohol,  72 ;  ammonia- 
carmine,  173  ;  picro-carmine,  171, 
172 ;  quinoleiu  blue,  223  ;  pur- 
purin,  264 ;  impregnation,  secon- 
dary, 242 ;  impregnation,  with 
silver,  243—245  ;  with  gold  and 
formic  acid,  252;  with  gold  and 
lemon  juice,  252 ;  after  blacken- 
ing of  gold,  258 ;  iodised  serum, 
270  ;  injections,  carmine,  300 ;  in- 
jections, Prussian  blue,  303,  309; 
impregnation  injections,  306 ; 
maceration,  313,  316,  317,  318; 
decalcification,  324 ;  tactile  cor- 
puscles, 368  ;  cornea,  369 ;  nerve 
and  muscle,  372;  corpuscles  of 
Golgi,  373  ;  bladder  of  frog,  375  ; 
medullated  nerve,  395 ;  retina, 
431,  433  ;  areolar  tissue,  435  ; 
clasmatocytes,  440 ;  bone,  442 ; 
goblet-cells,  456 ;  glands,  457. 

RAWITZ,  picro-uitro-chromic  acid,  69  • 
picro-nitro-osmic  acid,  69  ;  carrna- 
lum,  167  ;  inversion  stains,  226  ; 
artificial  alizarin,  227  ;  bleaching, 
329;  mucicarminic  acid,  455; 
eyes  of  Lamellibranchs,  463. 

Reagents,  161. 

RECKLINGHATJSEN,  silver  impregna- 
tion, 245,  247. 

Reconstruction  from  sections,  332 ; 
and  see  Orientation. 

REDDING,  gold  impregnations,  258. 

REDENBAUGH,  narcotisation,  16. 

REES,  VAN,  larvre  of  Diptera,  351. 

Refraction,  indices  of,  80. 

REGATTD,  silver  impregnation,  246. 

Regressive  staining,  155, "  196 ;  with 
tar  colours,  general  directions, 
196—200. 

REHM,  benzin  colophonium,  286 ; 
stains  for  nerve-cells,  392,  410. 

REICH,  silver  impregnation,  245. 

REICHENBACH,  ova  of  Astacus,  353. 

REINBACH,  triacid  mixture,  219. 

REINHOLD-GILTAY,  microtome,  89. 

REINKE,  gentian  and  orange  stain, 
214;  lysol,  318;  horny  tissues, 
368. 


REJSEK,  corrosion,  322. 
REMAK,  hardening  ova,  343. 
RENATJT,  hsematoxyliu  and  eosin,  266  ; 

silver  staining,  246. 
Reptilia,   embryology,    340  ;    nervous 

centres,  387. 
RESEGOTTI,  staining  by  substitution, 

198;  safranin,  202,  204. 
Resins  and  balsams,  281. 
Retina,  19,  431,  et  seq. 
RETTEEEE,  embryology  of  rabbit,  335 ; 

smooth  muscle,  374. 
RETTEEEE  and  ZELLNEB,  natural  in- 
jections, 311. 

RETZITJS,  methylen  blue,  233,  235. 
REZZONICO,  medullated  nerve,  395. 
RHUMBLEE,  paraffin  imbedding,  94; 

methyl  green  and  eosin,  221. 
RIBBEET,     phospho-molybdic     ha3ma- 

toxylin,    191 ;    connective   tissue, 

436. 

!  Ribbon  section-cutting,  112. 
I   RICHAED,  narcotisation,  15. 

RIEDEE,  fat,  437. 
|  RIEVEL,  Ophryotrocha,  471. 
Ringing  wet  mounts,  289. 
RIPAET  and  PETIT'S  liquid,  65,  273. 
Ripening  of  hsematoxylin,  177 — 179. 
RITTEE,  Ascidians,  347  ;  ova  of  Chiro- 

nomus,  351. 
ROBEET,  Aplysia,  462. 
ROBEETSON,  imbedding  method,  135  ; 

nerve  stain,  404,  406;   platinum 

impregnation,  428. 
ROBIN,    injections,     298—300,     306 ; 

natural  ditto,  311. 

j   ROBINSKI,  silver  impregnation,  245. 
ROLLETT,  freezing  method,  139 ;  cor- 
nea, 316,  370. 

ROOSEVELT,  pyrogallate  of  iron,  260. 
ROSE,  bone,  443. 
Rose  B  a  1'eau,  221. 
Rose  de  naphthalin,  207. 
ROSENSTADT,  fixing  liquid,  60  ;  eyes  of 

Decapods,  468. 
ROSIN,  "  neutral  "  dyes,  194 ;  neutral 

red,  221,  392. 

Rossi,  nerve  stain,  404;  blood,  449. 
ROSSOLIMOW  and  BUSCH,  nerve  stain, 

405. 


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